Crystal

ABSTRACT

The present invention relates to a crystal. In particular the present invention relates to a crystal of the N-domain of ACE protein. The present invention also relates to methods, processes, domain specific modulators, pharmaceutical compositions and uses of the N-domain crystal and the structure co-ordinates thereof.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/160,562, filed Nov. 20, 2008, (now U.S. Pat. No. 8,796,006),incorporated herein by reference in its entirety, which is the U.S.national phase of International Patent Application No.PCT/GB2007/000064, filed Jan. 10, 2007, incorporated here by referencein its entirety, which claims the benefit of priority of Great BritainPatent Application No. 0600406.3, filed Jan. 10, 2006.

Incorporated herein by reference in their entirety are computer-readableforms of a Sequence Listing and Tables A and B submitted concurrentlyherewith and identified as follows: ASCII text file named“44091A_SubSeqListing.txt”; 42,018 bytes; created Apr. 5, 2016 and“Table A.txt”‘ 1,470,646 bytes, created May 19, 2014, respectively.

LENGTHY TABLES The patent contains a lengthy table section. A copy ofthe table is available in electronic form from the USPTO web site(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US09434933B2). Anelectronic copy of the table will also be available from the USPTO uponrequest and payment of the fee set forth in 37 CFR 1.19(b)(3).

FIELD OF INVENTION

The present invention relates to a crystal. In particular the presentinvention relates to a crystal of the N-domain of ACE protein.

The present invention also relates to methods, processes, domainspecific modulators, pharmaceutical compositions and uses of theN-domain crystal and the structure co-ordinates thereof.

BACKGROUND TO THE INVENTION

Angiotensin-converting enzyme (peptidyl dipeptidase A, EC 3.4.15.1, ACE)is a zinc-dependent dipeptidyl carboxypeptidase with diversephysiological functions, including that of blood pressure regulation viaangiotensin II production and bradykinin inactivation. Somatic ACE(sACE), a type I transmembrane protein, is composed of two homologouscatalytic domains (N- (SEQ ID NO: 1) and C-domains) arising from a geneduplication event (Soubrier et al., 1988). The germinal form of ACE(testis ACE, (tACE) SEQ ID NO: 2) (Ehlers et al., 1989) originates fromthe same gene as sACE, but has a tissue-specific promoter located withinintron 12. Testis ACE (SEQ ID NO: 2) plays a crucial role inreproduction (Hagaman et al., 1998)

Despite sharing˜60% sequence identity with the C-domain, the N-domainhas its own distinctive physicochemical and functional properties. It isthermally more stable than the C-domain (Voronov et al., 2002), moreresistant to proteolysis under denaturing conditions and is lessdependent on chloride activation relative to the C-domain (Wei et al.,1991; Jaspard et al., 1993). The N- and the C-domains are joined by alinker that is susceptible to proteolysis (Sturrock et al., (1997),Biochem. Biophys. Res. 236, 16-19). It has also been suggested that theN- and the C-domains have unique physiological roles and that they havenegative effect on each other (Woodman et al., 2005).

Substrates such as the hemoregulatory peptide AcSDKP (Rousseau et al.,1995), angiotensin 1-7 (Deddish et al., 1998), and the enkephalinprecursor Met⁵-Enk-Arg⁶-Phe⁷ (Deddish et al., 1997) are specific for theN-domain, whereas the physiological substrates bradykinin andangiotensin I are hydrolysed with similar catalytic efficiency ascompared with the C-domain.

It has been reported that the N-domain preferentially hydrolyses the Abeta peptide of the amyloid precursor protein resulting in inhibition ofA beta aggregation and cytotoxicity (Oba et al., 2005). The widely-usedACE inhibitor captopril shows modest selectivity for the N-domain (Weiet al., 1992); however, the phosphinic peptide inhibitor RXP-407 has adissociation constant three orders of magnitude lower for the N-domainof the enzyme (Dive et al., 1999).

The N-domain has 10 N-linked sites of which 7 are unique to theN-domain. The different glycan profile of the N-domain is likelyresponsible for the carbohydrate-mediated dimerisation of sACE which hasbeen described under certain conditions (Kost et al., 2003). Moreover,the difference in glycosylation could impact on the structural basis forepitope recognition and epitope mapping of the N-domain has revealed aregion that might play a role in the relatively inefficient ectodomainshedding of sACE compared to its germinal isoform (Balyasnikova et al.,2005).

Both the N- and the C-domains of ACE protein are heavily glycosylated innature, a feature that has hampered 3D structural determination of theprotein and of each of the domains.

We previously described the 3D structure of the ACE protein(International Patent Application PCT/GB03/03966 (published as WO04/024765). This 3D structure was that of the underglycosylated C-domainof ACE protein.

This underglycosylated 3D structure, however, provides limitedinformation on the structure of the N-domain of ACE, nor is it ideal forscreening or designing domain specific modulators suitable forpharmaceutical use nor indeed for studying the functional interactionbetween the N- and the C-domains of ACE protein.

Therefore there is a need to obtain a crystal of the N-domain of ACEprotein with sufficient quality to allow crystallographic data to beobtained. Further, there is a need for such a crystal to allow thedetermination of the crystal structure of the N-domain of ACE. Finallythere is a need for procedures for studying the interplay between the N-and the C-domains and screening for domain specific modulators using the3D structural information of the N-domain of ACE protein.

SUMMARY OF THE INVENTION

The present inventors have now for the first time been able to describethe three-dimensional structure of the N-domain (SEQ ID NO: 1) of sACEprotein.

According to a first aspect of the present invention there is provided acrystal of the N-domain (SEQ ID NO: 1) of ACE protein. Preferably theN-domain of ACE protein is minimally glycosylated.

Preferably, the N-domain of ACE protein is minimally glycosylated byincorporating one or more glycosylation sites and/or one or morepartially glycosylated sites. More preferably, the minimallyglycosylated N-domain of ACE protein is minimally glycosylated at aminoacids 25, 45, 117, 289 and 480 of SEQ ID NO: 1.

Preferably the N-domain of ACE protein comprises an inter-domain linkerregion. This linker region joins the N- and the C-domains and has beenreported to be susceptible to proteolysis (Sturrock et al., (1997),Biochem. Biophys. Res. 236, 16-19). No one has previously visualisedthis linker region in a 3D structure. No one has previously analysed thephysiological role of this linker region and no one has previouslystudies the role of the linker in the functional interplay between theN- and the C-domains based on 3D structural data.

Preferably, the crystal of the N-domain of ACE protein (SEQ ID NO: 1)comprises atoms arranged in a spatial relationship represented by atleast a portion of the structural co-ordinates set forth in Table A (SEQID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8).

Preferably, the crystal belongs to the space group C222₁.

Preferably, the crystal has the unit cell dimensions: a=101.12 Å,b=211.32 Å, c=171.27 Å.

Preferably, the crystal is a crystal of the N-domain of human ACEprotein (SEQ ID NO: 1).

Preferably, the crystal of the N-domain of ACE protein comprises aligand bound to the N-domain of ACE protein or a portion thereof. Morepreferably the ligand modulates the activity of the N-domain of ACEprotein. More preferably the ligand is an inhibitor of the N-domain ofACE protein such as lisinopril or a derivative thereof. More preferablythe crystal of the N-domain of ACE protein comprising lisinoprilcomprises atoms arranged in a spatial relationship represented by atleast a portion of the structural co-ordinates set forth in Table B (SEQID NOs: 7 and 8).

Preferably the crystal of the N-domain of ACE protein comprising theinhibitor lisinopril has the unit cell dimensions of: a=101.32 Å,b=211.90 Å, c=171.03 Å.

In a second aspect, the present invention relates to a method ofpreparing a crystal of the N-domain of ACE protein comprising the stepsof: (a) culturing host cells expressing N-domain of ACE protein; (b)purifying the N-domain of ACE protein; and (c) crystallising theN-domain of ACE protein.

Preferably, the N-domain of ACE protein is crystallised using about 5 mMHEPES and about 0.1 mM PMSF with an equal volume of a reservoir solutioncontaining about 0.2M lithium sulphate, about 15% PEG 4000, about 100 mMCH₃COONa.3H₂O pH 4.9 and about 10 μM ZnSO₄.7H₂O.

Preferably, the crystal that is prepared has a structure defined by atleast a portion of the structural co-ordinates set forth in Table A (SEQID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8).

Preferably, the crystal belongs to the space group C222₁.

Preferably, the crystal has the unit cell dimensions: a=101.12 Å,b=211.32 Å, c=171.27 Å.

Preferably, the N-domain of ACE protein is the N-domain of human ACEprotein (SEQ ID NO:1).

Preferably, the N-domain of ACE protein is crystallised in the presenceof a ligand. More preferably the ligand is a modulator of the N-domainof ACE protein. More preferably the ligand is an inhibitor of theN-domain of ACE protein such as lisinopril or a derivative thereof. Morepreferably the crystal of the N-domain of ACE protein comprisinglisinopril comprises atoms arranged in a spatial relationshiprepresented by at least a portion of the structural co-ordinates setforth in Table B (SEQ ID NOs: 7 and 8).

Preferably the crystal of the N-domain of ACE protein comprising theinhibitor lisinopril has the unit cell dimensions of: a=101.32 Å,b=211.90 Å, c=171.03 Å.

Preferably the crystal of the N-domain of ACE protein that is preparedin the presence of a ligand is crystallised using about 5 mM HEPES andabout 0.1 mM PMSF with an equal volume of a reservoir solutioncontaining about 0.2M lithium sulphate, 18% PEG 4000, about 100 mMCH₃COONa.3H₂O pH 4.9 and about 10 μM ZnSO₄.7H₂O.

In a third aspect, the present invention relates to a method ofscreening for a modulator of the N-domain of ACE protein wherein themethod comprises the use of a crystal according to the presentinvention. Preferably, the screening method comprises the steps of: (a)providing at least a portion of the structural co-ordinates set forth inTable A (SEQ ID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8); (b)employing at least a portion of the structural co-ordinates set forth inTable A (SEQ ID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8) to designor select or synthesise a putative modulator of the N-domain of ACEprotein; (c) contacting the putative modulator of the N-domain with theN-domain or a mutant, variant, homologue, derivative or fragment thereofin the presence of a substrate; and (d) screening the putative modulatorof the N-domain of ACE protein in an assay for the potential to modulatethe N-domain.

Comparisons of the 3D structures of the N-domain of ACE protein andC-domain of ACE protein show that the two domains are structurally verysimilar.

Therefore, according to a fourth aspect, the present invention relatesto a method of screening for a modulator of the C-domain of ACE proteinwherein the method comprises the use of a crystal according to thepresent invention. Preferably, the screening method comprises the stepsof: (a) providing at least a portion of the structural co-ordinates setforth in Table A (SEQ ID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8);(b) employing at least a portion of the structural co-ordinates setforth in Table A (SEQ ID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8)to design or select or synthesise a putative modulator of the C-domainof ACE protein; (c) contacting the putative modulator of the C-domainwith the C-domain or a mutant, variant, homologue, derivative orfragment thereof; and (d) screening the putative modulator of theC-domain of ACE protein in an assay for the potential to modulate theC-domain.

Preferably, at least a portion of the structural co-ordinates set forthin Table A (SEQ ID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8) and/orthe putative modulator of the N-domain of ACE protein or C-domain of ACEprotein and/or the substrate are provided on a machine-readable datastorage medium comprising a data storage material encoded with machinereadable data.

Preferably, the putative N-domain of ACE protein modulator or C-domainof ACE protein modulator is selected from a library of compounds. Morepreferably, the putative N-domain of ACE protein modulator or C-domainof ACE protein modulator is selected from a database. More preferably,the putative N-domain of ACE protein modulator or C-domain of ACEprotein modulator is designed de novo. More preferably, the putativeN-domain of ACE protein modulator or C-domain of ACE protein modulatoris designed from a known ACE modulator. More preferably, the design orselection of the putative N-domain of ACE protein modulator or C-domainof ACE protein modulator is performed in conjunction with computermodelling.

Preferably, the putative N-domain of ACE protein modulator or C-domainof ACE protein modulator is useful in the prevention and/or treatment ofACE related disorder. More preferably, the ACE related disorder ishypertension, myocardial infarction or congestive heart failure.

In a fifth aspect, the present invention relates to a process comprisingthe steps of: (a) performing the method according to the third aspectand/or the fourth aspect of the present invention; (b) identifying oneor more modulators of the N-domain and/or C-domain; and (c) preparing aquantity of those one or more N-domain modulators and/or C-domainmodulators.

In a sixth aspect, the present invention relates to a process comprisingthe steps of: (a) performing the method according to the third aspectand/or the fourth aspect of the present invention; (b) identifying oneor more N-domain modulators and/or C-domain modulators; and (c)preparing a pharmaceutical composition comprising those one or moreN-domain modulators and/or C-domain modulators.

In a seventh aspect, the present invention relates to a processcomprising the steps of: (a) performing the method according to thethird aspect and/or the fourth aspect of the present invention; (b)identifying one or more N-domain modulators and/or C-domain modulators;(c) modifying those one or more N-domain modulators and/or C-domainmodulators; and (d) optionally preparing a pharmaceutical compositioncomprising those one or more N-domain modulators and/or C-domainmodulators.

In an eighth aspect, the present invention relates to a method ofobtaining structural information about a molecule or a molecular complexof unknown structure by using at least a portion of the structureco-ordinates of the N-domain of ACE protein, comprising the steps of:(a) generating X-ray diffraction data from a crystallised molecule ormolecular complex; (b) applying at least a portion of the structureco-ordinates of the N-domain of ACE protein to said X-ray diffractionpattern to generate a three dimensional electron density map of at leasta portion of the molecule or molecular complex; and (c) using all or aportion of the structure co-ordinates of the N-domain of ACE protein togenerate homology models of the N-domain of ACE protein. Preferably thestructural information of the structure co-ordinates of ACE protein areused for domain co-operativity studies.

In a ninth aspect, the present invention relates to a modulator of theN-domain of ACE protein and/or the C-domain of ACE protein identified bythe method according to the third aspect and/or the fourth aspect of thepresent invention. Preferably, the modulator inhibits the N-domain ofACE protein and/or the C-domain of ACE protein.

In a tenth aspect, the present invention relates to a pharmaceuticalcomposition comprising an N-domain of ACE protein modulator and/or aC-domain of ACE protein modulator according to the ninth aspect of thepresent invention and a pharmaceutically acceptable carrier, diluent,excipient or adjuvant or any combination thereof.

In an eleventh aspect, the present invention relates to a method ofpreventing and/or treating an ACE related disorder comprisingadministering a modulator of the N-domain of ACE protein and/or theC-domain of ACE protein according to the ninth aspect of the presentinvention and/or a pharmaceutical composition according to the tenthaspect of the present invention, wherein said modulator of the N-domainof ACE protein and/or the C-domain of ACE protein and/or saidpharmaceutical composition is capable of causing a beneficialpreventative and/or therapeutic effect.

In a twelfth aspect, the present invention relates to a computer forproducing a three-dimensional representation of the N-domain of ACEprotein wherein said computer comprises: (a) a computer-readable datastorage medium comprising a data storage material encoded withcomputer-readable data, wherein said data comprises the structureco-ordinates of the N-domain of ACE protein; (b) a working memory forstoring instructions for processing said computer-readable data; (c) acentral-processing unit coupled to said working memory and to saidcomputer-readable data storage medium for processing saidcomputer-machine readable data into said three-dimensionalrepresentation; and (d) a display coupled to said central-processingunit for displaying said three-dimensional representation.

In a thirteenth aspect, the present invention relates to amachine-readable data storage medium comprising a data storage materialencoded with machine-readable data, wherein the data is defined by atleast a portion of the structural co-ordinates of the N-domain (SEQ IDNO: 1) of ACE protein set forth in Table A (SEQ ID NOs: 5 and 6) orTable B (SEQ ID NOs: 7 and 8).

In a fourteenth aspect, the present invention relates to the use of anN-domain of ACE protein crystal in the preparation of a medicament toprevent and/or treat an ACE related disorder. Preferably, the ACErelated disorder is hypertension, myocardial infarction or congestiveheart failure.

In a fifteenth aspect, the present invention relates to the use of atleast a portion of the structural co-ordinates set forth in Table A (SEQID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8) to screen formodulators of the N-domain of ACE protein and/or C-domain of ACEprotein.

In a sixteenth aspect, the present invention relates to the use of atleast a portion of the structural co-ordinates set forth in Table A (SEQID NO: 5 and 6) or Table B (SEQ ID NOs: 7 and 8) to solve the structureof the crystalline form of any other domain with significant amino acidsequence homology to any functional domain of the N-domain of ACEprotein.

In a seventeenth aspect, the present invention relates to the use of atleast a portion of the structural co-ordinates set forth in Table A (SEQID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8) in molecular designtechniques to design, select and synthesise modulators of the N-domainof ACE protein and/or C-domain of ACE protein.

In an eighteenth aspect, the present invention relates to the use of atleast a portion of the structural co-ordinates set forth in Table A (SEQID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8) in the development ofcompounds that can isomerise to reaction intermediates in the chemicalreaction of a substrate or other compound that binds to the N-domain ofACE protein and/or the C-domain of ACE protein.

In a nineteenth aspect, the present invention relates to the use of atleast a portion of the structural co-ordinates set forth in Table A (SEQID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8) to screen smallmolecule databases for chemical entities or compounds that modulate theN-domain of ACE protein and/or C-domain of ACE protein.

In a twentieth aspect, the present invention relates to the use of atleast a portion of the structural co-ordinates set forth in Table A (SEQID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8) to solve the structureof the crystalline form of any other domain with significant amino acidsequence homology to any functional domain of the N-domain of ACEprotein. Preferably, the structure of the crystalline form of any otherdomain with significant amino acid sequence homology to any functionaldomain of the N-domain of ACE protein is solved using molecularreplacement.

In a twenty first aspect, the present invention relates to the use of atleast a portion of the structural co-ordinates set forth in Table A (SEQID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8) to identify thebinding site of any modulator of the N-domain of ACE protein. Preferablythe binding site is that of lisinopril or derivatives thereof.

DETAILED DESCRIPTION OF THE INVENTION

N-Domain of ACE Protein

ACE (EC 3.4.15.1) is a membrane associated peptidyl dipeptidase.

There are two tissues specific isoforms of ACE protein differentiallytranscribed from the same gene.

Somatic ACE (sACE), a type I transmembrane protein, is composed of twohomologous catalytic N- (SEQ ID NO: 1) and C-domains, arising from agene duplication event (Soubrier et al., 1988).

Germinal ACE (testis ACE, (tACE) (SEQ ID NO: 2)) has a tissue-specificpromoter located within intron 12 (Ehlers et al., 1989).

Despite sharing˜60% sequence identity with the C-domain, the N-domain(SEQ ID NO: 1) has its own distinctive physicochemical and functionalproperties. It has also been reported that the two domains displaynegative co-operativity effect on each other (Woodman et al., 2005). Inthe present invention, co-operativity studies between the N- (SEQ IDNO: 1) and C-domains of ACE protein is based on studies carried out byGuy et al., 2003 (Biochemistry, 42:13185-13192) and Towler et al., 2004(The Journal of Biological Chemistry, 17:17996-18007). In their studies,the authors compare ACE structures with those of the ACE2 (SEQ ID NO: 3)homologue that more closely resembles the N-domain (SEQ ID NO: 1) of theACE protein. See FIG. 4 below.

As used herein the term “negative co-operativity” refers to the capacityof the two domains of ACE protein to modulate the biochemical activitiesof each other. By way of example the N-domain may cause steric hindranceof the C-domain by preventing substrate from accessing the active siteof the C-domain and vice versa the C-domain may cause steric hindranceof the N-domain by preventing substrate from accessing the active siteof the N-domain.

The N- and the C-domains are heavily glycosylated, which has hamperedthe determination of the 3D structure of the ACE protein.

Previously we described the 3D structure of the C-domain ofundeglycosylated ACE protein (see International Patent ApplicationPCT/GB03/03966 (published as WO 04/024765)).

The present inventors have now been able to obtain a minimallyglycosylated N-domain of ACE protein.

As used herein the term “minimally glycosylated” means that one or moreoligosaccharide chains are linked to one or more amino acid residues inthe N-domain of ACE protein. As used herein the term “oligosaccharide”refers to a carbohydrate molecule which comprises less than 10 sugarmolecules where the sugar molecules can be any one or more ofmono-saccharides, di-, tri-, and/or tetra-saccharides.

As used herein, the term “N-domain of ACE protein” includes allvertebrate and mammalian forms of the N-domain of ACE protein and isintended to cover mutants, variants, homologues, derivatives andfragments thereof. Preferably, the mutants, variants, homologues,derivatives and fragments thereof have the activity of the N-domain ofACE protein. Preferably the N-domain of the ACE protein is minimallyglycozylated.

Crystal

In one aspect of the present invention, there is provided a crystalstructure of the N-domain of ACE protein in its minimally glycozylatedform and its ligand-bound form.

As used herein, the term “crystal” means a structure (such as a threedimensional (3D) solid aggregate) in which the plane faces intersect atdefinite angles and in which there is a regular structure (such asinternal structure) of the constituent chemical species. Thus, the term“crystal” can include any one of: a solid physical crystal form such asan experimentally prepared crystal, a 3D model based on the crystalstructure, a representation thereof—such as a schematic representationthereof, a diagramatic representation thereof, or a data set thereof fora computer.

The crystals of the present invention may be prepared by purifying theN-domain of ACE protein and then crystallising the purified protein. TheN-domain of ACE protein may also be prepared by expressing a nucleotidesequence encoding the N-domain of ACE protein in a suitable host cell.

In one preferred embodiment, the N-domain of ACE crystal comprises theinter-domain linker region.

The N-domain of ACE protein may be purified using various methods knownto a person skilled in the art, for example, from conditioned media byaffinity chromatography on a Sepharose-28-lisinopril affinity resin (Yuet al. 1997). The protein may be quantified by amino acid analysis andassayed for activity using the substrate hippuryl-L-histidyl-L-leucine,as described previously (Ehlers, M R E, Chen, Y -N, Riordan, J F (1991)Proc. Natl. Acad. Sci. USA 88, 1009-1013).

The purified N-domain of ACE proteins may be stored at −20° C. in 5 mMHEPES and 0.1 mM PMSF.

Concentration may be performed with the aid of a filtration system andthe protein concentrate may be immediately used for crystallisationpurposes. The protein concentrate may be crystallised using, forexample, the vapour diffusion hanging drop method at a temperature offrom about 4° C. to about 30° C., preferably from about 8° C. to about20° C., more preferably from about 12° C. to about 18° C., mostpreferably at about 16° C. The crystallisation temperature may bedependent on the additives present in the protein solution.

Typically, the best crystals for the N-domain of ACE proteins are grownat a temperature in the range of from 10 to 20° C., preferably from 12to 18° C., most preferably at 16° C. by the vapour diffusion hangingdrop method by mixing 1 μl of the protein solution at about 4 mg/ml. Thesolution comprising HEPES in the range of from 1 to 10 mM, preferably inthe range of from 2 to 8 mM, more preferably in the range of from 4 to 6mM, most preferably 5 mM HEPES and PMSF in the range of from 0.025 to0.2%, preferably from 0.05 to 0.15%, more preferably from 0.075 to0.125, most preferably 0.1% PMSF with an equal volume of a reservoirsolution. The reservoir solution comprising lithium sulphate in therange of from 0.025 to 0.4M, preferably in the range of from 0.05 to0.35M, more preferably from 0.075 to 0.3M, most preferably containing0.2M lithium sulphate, PEG 4000 in the range of from 5% to 25%,preferably from 10% to 20%, most preferably from 15% to 18% PEG 4000,CH₃COONa.3H₂O in the range of from 50 to 150 mM, preferably from 75 to125 mM, more preferably from 90 to 110 mM, most preferably 100 mMCH₃COONa.3H₂O, wherein the CH₃COONa.3H₂O has a pH in the range of from3.9 to 5.9, preferably a pH from 4.5 to 5.5, more preferably a pH from4.7 to 5.2, most preferably a pH of 4.9 and ZnSO₄.7H₂O in the range offrom 5 to 20 μM, preferably from 7.5 to 12.5 μM, most preferably 10 μMZnSO₄.7H₂O.

Crystals usually appear within 1 to 2 weeks and grow to their maximumsize after about a month.

The present invention related to a crystal of the N-domain of ACEcomprising atoms arranged in a spatial relationship represented by atleast a portion of the structural co-ordinates set forth in Table A (SEQID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8).

Preferably, the crystal belongs to the space group C222₁ and unit celldimensions: a=101.12 Å, b=211.32 Å, c=171.27 Å.

Preferably, the N-domain of ACE protein is the N-domain of anyvertebrate and mammalian.

Preferably the N-domain of ACE protein is the N-domain of human ACEprotein (SEQ ID NO: 1).

Complexes may be obtained by growing the crystals in the presence of aligand—such as a test compound. In these experiments the proteinsolution is mixed with the ligand and an equal volume of the reservoirsolution before setting up the crystallisation. Single crystals suitablefor diffraction work typically appear after about 1-2 weeks.

Typically, the protein comprising the N-domain of ACE protein ispurified to homogeneity for crystallisation. Purity of the N-domain ofACE protein may be measured by typical techniques such as SDS-PAGE, massspectrometry and hydrophobic HPLC.

The structure of the crystals of the invention may contain aportion—such as at least 25%, at least 50%, at least 75%, or preferablyat least 90%, at least 95%, at least 98%, or at least 99%—of theco-ordinates set forth in Table A (SEQ ID NOs: 5 and 6) or Table B (SEQID NOs: 7 and 8). Preferably, the crystal structure of the inventioncontains all of the co-ordinates set forth in Table A (SEQ ID NOs: 5 and6) or Table B (SEQ ID NOs: 7 and 8)

Preferably, the crystals are usable in X-ray crystallography techniques.

Preferably, the crystals used can withstand exposure to X-ray beams usedto produce diffraction pattern data necessary to solve the X-raycrystallographic structure.

Preferably, prior to data collection, the crystals are flash-cooled atabout 100 K in a cryoprotectant. The cryoprotectant comprising lithiumsulphate in the range of from 0.025 to 0.4M, preferably in the range offrom 0.05 to 0.35M, more preferably from 0.075 to 0.3M, most preferablycontaining 0.2M lithium sulphate, PEG 4000 in the range of from 5% to25%, preferably from 10% to 20%, most preferably from 15% to 18% PEG4000, CH₃COONa.3H₂O in the range of from 50 to 150 mM, preferably from75 to 125 mM, more preferably from 90 to 110 mM, most preferably 100 mMCH₃COONa.3H₂O, wherein the CH₃COONa.3H₂O has a pH in the range of from3.9 to 5.9, preferably a pH from 4.5 to 5.5, more preferably a pH from4.7 to 5.2, most preferably a pH of 4.9 and ZnSO₄.7H₂O in the range offrom 5 to 20 μM, preferably from 7.5 to 12.5 μM, most preferably 10 μMZnSO₄.7H₂O.

The X-ray data may be collected at a Synchrotron Radiation Source.Preferably, the X-ray data are collected at a Synchrotron RadiationSource at 100° K.

Preferably, the crystal has a resolution determined by X-raycrystallography of about 3.5 Å or less, more preferably a resolution ofabout 3.0 Å or less, more preferably a resolution of about 2.8 Å orless, more preferably, a resolution of about 2 Å or less, morepreferably, a resolution of about 1.5 Å or less, most preferably, 1 Å orless.

Glycosylation of the N-Domain of Ace Protein

Many proteins in eukaryotic cells are glycoproteins that containoligosaccharide chains covalently linked to certain amino acids.Glycosylation is known to affect protein folding, interaction betweenprotein domains, localisation and trafficking, protein solubility,antigenicity, biological activity and half-life, as well as cell-cellinteractions.

Protein glycosylation can be divided into four main categories mainlydepending on the linkage between the amino acid and the sugar molecule.These are N-linked glycosylation, O-linked glycosylation,C-mannosylation and GPI anchor attachments. N-glycosylation ischaracterised by the addition of a sugar to the amino group of anasparagine.

For N-glycosylation, the sequence motif Asn-Xaa-Ser/Thr (wherein Xaa isany amino acid other than Pro) has been defined as a prerequisite forglycosylation. Although rare, the sequence motif Asn-Xaa-Cys can also bean acceptor site. N-glycans can be subdivided into three distinct groupscalled ‘high mannose type’, ‘hybrid type’, and ‘complex type’, with thecommon pentasaccharidecore—Manp(alpha1,6)-(Manp(alpha1,3))-Manp(beta1,4)-GlcpNAc(beta1,4)GlcpNAc(beta1,N)-Asn—occurring in all three groups. The relationshipbetween all three types can be ascribed to the fact that they originatefrom one precursor oligosaccharide which contains the described commonpentasaccharide core Man3-GlcNAc2, and some additional sugar residuesand the non-reducing end, and is then processed enzymatically to yieldthese three types. Since the hydroxyl group of Ser/Thr is thought to beinvolved in hydrogen bonding during the enzymatic attachment of theoligosaccharide precursor molecule to yield a favourable conformationfor the action of the oligosaccharyltransferase, it has been suggestedfor proline that the steric hindrance might be too large (Kornfeld(1985) Ann. Rev. Biochem. 54: 631-64), preventing glycosylation at Procontaining sites. The negative influence of aspartic acid towardsglycosylation can be ascribed to the negative charge on the side chainof this residue. In addition some cases have been reported where Ser/Thris replaced by cysteine. While Ser replacement by Cys generally leads todecreased glycosylation, it has been shown (Kasturi 1995 J. Biol. Chem.270: 14756-61) that substitution by Thr at a given potentialglycosylation site can lead to increased glycosylation. This is inaccordance with the model of hydrogen bonding being an important factorduring the attachment of the precursor molecule to the protein. Althoughthere are usually many potential glycosylation sites in a protein it hasbeen estimated that glycosylation occurs only at one third of them.Mostly at those sites where the surrounding amino acids allow theformation of a beta turn.

Various glycoforms of ACE have been described. By way of example,Sadhukhan & Sen disrupted specific glycosylation sites in rabbit tACE toelucidate the glycosylation requirements for the expression andprocessing of active testis ACE. There are five potential N-linkedglycosylation sites in the rabbit tACE sequence, with an additional sixin the somatic form. A null mutant, where all five sites had beendisrupted, behaved similarly to wild-type tACE expressed in the presenceof the glycosylation-inhibitor, tunicamycin. It was degradedintracellularly and failed to be detected in culture medium, confirmingprevious findings that tACE requires N-linked glycosylation to beexpressed in an active form. Expression of the remaining mutants showeda preference for N-linked glycosylation at the N-terminus and that thepresence of sugars at a single N-terminal site was necessary andsufficient to produce enzymatically-active tACE that was solubilised.The presence of glycosylation is not site-specific, as mutants that haveeither the first site or second site intact are expressed and active.However, glycosylation at the third site alone is not sufficient toproduce active protein in HeLa cells, albeit this mutant was expressedin yeast, indicating that the requirements for glycosylation arecell-specific.

N-linked glycosylation of human tACE (SEQ ID NO: 2) expressed in CHOcells at each site has been identified by MALDI-TOF mass spectrometry.There are seven potential N-linked sites in human tACE (SEQ ID NO: 2),five of which are complementary to the sites in rabbit tACE (7a). Theunique sites lie within the ectodomain (the fourth site) and in thejuxtamembrane stalk region, adjacent to the cleavage site (the seventhsite). As with the rabbit form, there appears to be a preference forglycosylation at the N-terminus as evidenced by MALDI-TOF massspectrometry of glycopeptides (Yu et al., 1997) and mutagenesis (Gordonet al., 2003) of glycosylation sites. Inhibition of complexoligosaccharide formation using a glucosidase I inhibitorN-butyldeoxynojirimycin (NB-DNJ) led to the production of an activeglycoform that was electrophoretically homogeneous.

There are ten putative N-glycosylation sites on the human N-domain (SEQID NO: 1) of ACE protein and Fourier difference density was observed atfive of these sites according to the crystal of the present invention.

Suitably, the crystal of the N-domain of ACE protein may compriseminimally glycosylated N-domain of ACE protein or a fragment thereof.For example, the minimally glycosylated N-domain of ACE protein maycomprise the sequence presented as (SEQ ID NO: 1).

To obtain a minimally glycosylated N-domain of ACE protein, variousmethods known to a person skilled in the art may be used. Both chemicaland enzymatic methods may be used. Hydrazinolysis of glycoproteins(Kuraya, N & Hase (1992) J Biochem (Tokyo) 112:122-126), is capable ofremoving both N- and O-linked sugars, although this results in thecomplete destruction of the protein component and is therefore notsuitable if recovery of the protein is desirable. More delicate chemicalmethods may be used such as trifluoromethanesulphonic acid (TFMS),however this method may also lead to a partial protein destruction.Alternatively, or in addition, other methods—such as site directedmutagenesis of glycosylated amino acids may also be employed.

Enzymatic methods which provide for partial sugar removal with noprotein degradation may also be used.

Use of the enzyme PNGase F is an effective method of removing mostN-linked oligosaccharides from glycoproteins (Tarentino & Plummer (1994)Methods in Enzymology, 230: 44-57). The oligosaccharide is left intactand therefore suitable for further analysis (the asparagine residue fromwhich the sugar is removed is deaminated to aspartic acid, the onlymodification to the protein).

Other commonly used endoglycosidases include Endoglycosidase H (Kobata(1979) Anal Biochem 100:1-14) and Endoglycosidase F (Trimble & Tarentino(1991) J. Biochem. 266:1646-1651). In a preferred method, the N-domainof ACE protein is digested with Endoglycosidase H (30 mU) in a suitablebuffer—such as 100 mM sodium phosphate, 0.1 mM ZnCl₂ and 1% BSA, pH 6.0for 16 h at 37° C. The endo H-treated protein is passed through a lectinaffinity column consisting of equal parts of concanavalin A, wheat germ,and lentil lectin, after equilibration with 20 mM Tris-HCl, 0.5 M NaClat pH 7.4. The minodeglycosylated ACE is collected in the flowthrough.Free oligosaccharides and any other impurities are removed from theflowthrough fraction by a final lisinopril-Sepharose affinitychromatography step. The homogeneity of the ACE protein afterdeglycosylation is confirmed by SDS-PAGE on a 4-20% acrylamide gel andMALDI-TOF mass spectrometry.

By way of example, a minimally glycosylated N-domain of ACE protein canbe obtained by incubating suitable host cells such as CHO cells untilconfluent and then substituting the growth medium with medium comprising1% FCS, 0.05% albumax I (Gibco BRL), 20 μM MSX and 1.5 mM of theglucosidase I inhibitor N-butyldeoxynojirimycin (NB-DNJ) (TorontoResearch Chemicals Inc.B691000 Lot 14-EG-91-1 and B691000 12-Cf-146-2).

Preferably, the crystal of the N-domain of ACE protein comprisesglycosylated N-domain of ACE protein or a fragment thereof. Morepreferably, the crystal of the N-domain of ACE protein comprises aminimally glycosylated N-domain of ACE protein or a fragment thereof.More preferably, the crystal of the N-domain of ACE protein comprisesN-glycosylation of asparagine residues. More, preferably the asparagineresidues are N-glycosylated with high mannose oligosaccharides.

Preferably, one or more of the asparagine residues of the N-domain ofACE protein can be N-glycosylated. More preferably, one or more of theasparagine residue of SEQ ID NO:1 are N-glycosylated. More, preferablyone or more of the asparagine residues 25, 45, 117, 289 and 480 of SEQID NO:1 can be N-glycosylated.

Preparing a Crystal of the N-Domain of Ace Protein

In another aspect, the present invention relates to a method ofpreparing a crystal of the N-domain of ACE protein, comprising the stepsof (a) culturing host cells comprising N-domain of ACE protein; (b)purifying the N-domain of ACE protein; and (c) crystallising theN-domain of ACE protein.

Preferably, the N-domain of ACE protein comprises an inter-domain linkerregion.

The N-domain of ACE protein may be purified using the methods describedherein.

Preferably, the N-domain of ACE protein is crystallised in the presenceof a ligand, for example, a modulator of the N-domain of ACE protein.

Modulators

The role of ACE protein in the pathogenesis of hypertension has resultedin an intensive search for modulators (eg. inhibitors) of the enzymethat could act as antihypertensive drugs (eg. U.S. Pat. No. 3,891,616,U.S. Pat. No. 3,947,575, U.S. Pat. No. 4,052,511 and U.S. Pat. No.4,053,651). Therapeutic vasodepressors—such as the compound captopril(D-2-methyl-3-mercaptopropanoyl-L-proline)—have been synthesised as ACEinhibitors. Numerous synthetic peptide derivatives have also been shownto be ACE inhibitors as disclosed in U.S. Pat. No. 3,832,337.

Natural substances that inhibit ACE include snake venom and thosederived from foodstuffs—such as ACE inhibiting peptides produced byenzymatic hydrolysis of proteins, such as casein or fish meat protein(by Hiroyuki Ukeda, Nippon Nogei Kagaku Kaishi (Journal of Japan Societyfor Bioscience, Biotechnology, and Agrochemistry, 66(1), 25-29 (1992)).

ACE inhibiting synthetic substances include captopril(D-2-methyl-3-mercaptopropanoyl-L-proline) which has already been put topractical application as an orally administered vasodepressor.

However, many currently used ACE inhibiting substances exhibit sideeffects in many cases and special attention needs to be exercised.

The present invention offers a novel concept in the field of ACE proteininhibitor design by providing conditions suitable for generatingspecific inhibitors to more precisely target and regulate the activityof the N- and/or C-domains of ACE protein. Preferably the modulators areN-domain of ACE protein specific and/or C-domain of ACE proteinspecific. These novel modulators can advantageously have reduced sideeffects.

The present invention provides the use of molecular design techniques todesign, select and synthesise chemical entities and compounds, includingACE modulating compounds, capable of binding to the N-domain of ACEprotein, in whole or in part.

Thus, in a further aspect, the present invention relates to a method ofscreening for a modulator of the N-domain of ACE protein wherein themethod comprises the use of a crystal of the N-domain of ACE protein.

Preferably, the method comprises the steps of: (a) providing at least aportion of the structural co-ordinates set forth in Table A (SEQ ID NOs:5 and 6) or Table B (SEQ ID NOs: 7 and 8); (b) employing at least aportion of the structural co-ordinates set forth in Table A (SEQ ID NOs:5 and 6) or Table B (SEQ ID NOs: 7 and 8) to design or select orsynthesise a putative modulator of the N-domain of ACE protein; (c)contacting the putative modulator of the N-domain of ACE protein withthe N-domain of ACE protein or a mutant, variant, homologue, derivativeor fragment thereof in the presence of a substrate; and (d) screeningthe putative modulator of the N-domain of ACE protein in an assay forthe potential to modulate the N-domain.

By way of example, the structure co-ordinates may be used to designcompounds that bind to the N-domain of ACE enzyme and may alter thephysical properties of the compounds (e.g. solubility) or the domain orthe enzyme itself. This invention may be used to design compounds thatact as modulators—such as competitive inhibitors—of the N-domain of ACEprotein by binding to all or a portion of the active site of theN-domain of ACE protein. Compounds may also be designed that act asnon-competitive inhibitors of the N-domain of ACE protein. Thesenon-competitive inhibitors may bind to all or a portion of the N-domainof ACE protein already bound to its substrate and may be more potent andspecific than known N-domain of ACE protein inhibitors that compete onlyfor the N-domain of ACE protein active site. Similarly, non-competitiveinhibitors that bind to and inhibit the N-domain of ACE protein whetheror not it is bound to another chemical ligand may be designed using thestructure co-ordinates of the N-domain of ACE protein as describedherein.

By way of example, it may be found that the COOH-binding active siteresidue differs between the N- and C-domain of ACE protein active sitesand/or that it may be amenable to the incorporation of a functionalitythat can covalently modify this residue to produce an irreversibledomain specific inhibitor design.

Due to the significant structural similarity between the N-domain andthe C-domain of ACE protein it is envisaged that the structuralco-ordinates of the crystal of the N-domain can also be used to designmodulators which are specific for the C-domain of ACE protein.

Accordingly, in a further aspect, the present invention relates to amethod of screening for a modulator of the C-domain of ACE proteinwherein the method comprises the use of a crystal of the N-domain of ACEprotein. Preferably, the method comprises the steps of: (a) providing atleast a portion of the structural co-ordinates set forth in Table A (SEQID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8); (b) employing atleast a portion of the structural co-ordinates set forth in Table A (SEQID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8) to design or select orsynthesise a putative modulator of the C-domain of ACE protein; (c)contacting the putative modulator of the C-domain of ACE protein withthe C-domain of ACE protein or a mutant, variant, homologue, derivativeor fragment thereof in the presence of a substrate; and (d) screeningthe putative modulator of the C-domain of ACE protein in an assay forthe potential to modulate the C-domain.

By way of example, the structure co-ordinates of the N-domain may beused to design compounds that bind to the C-domain of ACE enzyme and mayalter the physical properties of the compounds (e.g. solubility) or thedomains or the enzyme itself. This invention may be used to designcompounds that act as modulators—such as competitive inhibitors—of theC-domain of ACE protein by binding to all or a portion of the activesite of the C-domain of ACE protein. Compounds may also be designed thatact as non-competitive inhibitors of the C-domain of ACE protein. Thesenon-competitive inhibitors may bind to all or a portion of the C-domainof ACE protein already bound to its substrate and may be more potent andspecific than known C-domain of ACE protein inhibitors that compete onlyfor the C-domain of ACE protein active site. Similarly, non-competitiveinhibitors that bind to and inhibit the C-domain of ACE protein whetheror not it is bound to another chemical ligand may be designed using thestructure co-ordinates of the N-domain of ACE protein as describedherein. Advantageously, the specific modulators of the C-domain of ACEprotein do not adversely affect the enzymatic activity of the N-domainof ACE protein. By way of example, such C-domain specific inhibitors canpermit the N-domain to hydrolyse the vasodilator bradykinin and thusalleviate side effects such as persistent cough and angioedema.Accordingly, the C-domain specific modulators identified by thescreening methods described herein may have the capacity to overcomecertain side effects. By way of a further example, the C-domain specificinhibitors can also permit the hydrolysis of the N-domain specifichemoregulatory peptide AcSDKP.

In a preferred embodiment, at least a portion of the structuralco-ordinates set forth in Table A (SEQ ID NOs: 5 and 6) or Table B (SEQID NOs: 7 and 8) and/or the putative modulator of the N-domain of ACEprotein or C-domain of ACE protein and/or the substrate are provided ona machine-readable data storage medium comprising a data storagematerial encoded with machine readable data.

A crystal of the N-domain of ACE may be probed with a variety ofdifferent chemical entities or test compounds to determine optimal sitesfor interaction between modulators of the N-domain of ACE protein or theC-domain of ACE protein and the enzyme. For example, X-ray diffractiondata collected from crystals grown in the presence of chemical entitiesor test compounds may allow the elucidation of how the chemical entitiesor test compounds interact with the N-domain of ACE protein or theC-domain of ACE protein. Molecules that bind to those sites can then bedesigned and synthesised and tested for their capacity to modulate theactivity of the N-domain of ACE protein.

The present invention may also allow the development of compounds thatcan isomerise to reaction intermediates in the chemical reaction of asubstrate or other compound that bind to the N-domain of ACE protein orthe C-domain of ACE protein. Thus, the time-dependent analysis ofstructural changes in the N-domain of ACE protein during its interactionwith other molecules may be performed. The reaction intermediates of theN-domain of ACE protein or the C-domain of ACE protein may also bededuced from the reaction product in complex with the N-domain of ACEprotein. Such information is especially useful to design improvedanalogues of known N-domain or C-domain modulators or to design newN-domain or C-domain modulators based on the reaction intermediates andthe modulator complex. This may provide a new route for designingN-domain or C-domain of ACE protein modulators with high domainspecificity and stability. Preferably, this provides a new route fordesigning N-domain or C-domain of ACE protein modulators with highdomain specificity, high stability and low toxicity.

Small molecule databases or test compounds may be screened for chemicalentities or compounds that can bind in whole, or in part, to theN-domain or C-domain of ACE protein. Thus, in a preferred embodiment,the putative N-domain or C-domain modulator is selected from a libraryof compounds or a database. In this screening, the quality of fit ofsuch entities or compounds to the binding site may be judged by variousmethods—such as shape complementarity or estimated interaction energy(Meng, E. C. et al., J. Comp. Chem., 13, pp. 505-524 (1992)).

Because the N-domain of ACE protein or a mutant, variant, homologue,derivative or fragment thereof may crystallise in more than one crystalform, the structure co-ordinates of the N-domain of ACE protein, orportions thereof, may be particularly useful to solve the structure ofother crystal forms of the N-domain of ACE protein. They may also beused to solve the structure of the N-domain of ACE protein mutants,N-domain of ACE protein variants, N-domain of ACE protein homologues,N-domain of ACE protein derivatives, N-domain of ACE protein fragmentsand N-domain of ACE protein complexes.

Preferably, the structure co-ordinates of the N-domain of ACE proteinare used to solve the structure of the crystalline form of any otherprotein with significant amino acid sequence homology to any functionaldomain of the N-domain of ACE protein. By way of example, molecularreplacement may be used. In this method, the unknown crystal structure,whether it is another crystal form of the N-domain of ACE protein,N-domain of ACE protein mutant, N-domain of ACE protein variant,N-domain of ACE protein homologue (eg. another protein with significantamino acid sequence homology to any functional domain of the N-domain ofACE protein), N-domain of ACE protein derivative, N-domain of ACEprotein fragments or N-domain of ACE protein complex may be determinedusing the N-domain of ACE protein structure co-ordinates of the presentinvention. This method will provide a more accurate structural form forthe unknown crystal more quickly and efficiently than attempting todetermine such information ab initio.

Preferably, the structural co-ordinates of the N-domain of ACE proteinare used for domain interplay or also referred to as domainco-operativity studies. By way of example, molecular replacement may beused. In this method, unknown domain co-operativity with N-domain of ACEprotein, N-domain of ACE protein mutant, N-domain of ACE proteinvariant, N-domain of ACE protein homologue (e.g. another protein withsignificant amino acid sequence homology to the N-domain of ACEprotein), N-domain of ACE protein derivative, N-domain of ACE proteinfragments or N-domain of ACE protein complex may be determined using theN-domain of ACE protein structure co-ordinates of the present invention.This method will provide a more accurate structural form for the unknowncrystal more quickly and efficiently than attempting to determine suchinformation ab initio.

As used herein the term “domain co-operativity” refers to the capacityof each of the domains of ACE protein to modulate the biochemicalactivities of each other. As used herein the term “modulate” means toaffect, to vary, to adjust, to increase, to decrease or generally to beable to regulate or modify the biochemical activity of the N- orC-domain of ACE protein.

Preferably the structural co-ordinates of N-domain of ACE protein areused to study co-operativity between the N-domain of ACE protein and theC-domain of ACE protein.

In a preferred embodiment of the present invention, the crystal of theN-domain of ACE protein further comprises a ligand bound to the ACEprotein or a portion thereof. For example, the N-domain of ACE proteinmay be crystallised in a complex with a ligand that is an inhibitor ofthe N-domain of ACE protein e.g. lisinopril, captopril or RXP407.

By way of example the inhibitor is bound to the N-domain of ACE proteinor a portion thereof by contacting one of more residues of the N-domain(SEQ ID NO: 1) of ACE protein selected from: Gln259, Tyr369, Lys489 andTyr498.

The crystal structures of a series of such complexes may then be solvedby molecular replacement or in combination with MAD (MultiwavelengthAnomalous Dispersion) and/or MIRAS (Multiple Isomorphous Replacementwith Anomalous Scattering) procedures—and compared with that ofminimally glycosylated N-domain of ACE protein. Potential sites formodification within the binding sites of the N-domain may thus beidentified. This information provides an additional tool for determiningthe most efficient binding interactions, for example, increasedhydrophobic interactions, between the N-domain of ACE protein and aligand or a compound.

The information will also provide ideal conditions for designing domainspecific inhibitors to more precisely regulate the biochemical functionsof the N- and C-domains of ACE protein.

The structures and complexes of the N-domain of ACE protein may berefined using computer software—such as X-PLOR (Meth. Enzymol., vol. 114& 115, H. W. Wyckoff et al., eds., Academic Press (1985)), MLPHARE(Collaborative computational project Number 4. The CCP4 Suite: Programsfor Protein Crystallography (1994) Acta Crystallogr. D 50, 760-763) andSHARP [De La Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atomparameters refinement in the MIR and MAD methods (1997) Methods Enzymol.276, 472-494). Preferably, the complexes are refined using the programCNS (Brünger et al., (1998) Acta Crystallogr. D 54, 905-921). During thefinal stages of refinement water molecules, ions and inhibitor moleculesmay be inserted in the structure. This information may thus be used tooptimise known classes of modulators of the N-domain of ACE protein, eg.inhibitors, and more importantly, to design and synthesise novel classesof domain specific modulators.

The overall figure of merit may be improved by iterative solventflattening, phase combination and phase extension with the programSOLOMON (Abrahams, J. P. & Leslie, A. G. W. Methods used in structuredetermination of bovine mitochondrial F1 ATPase. (1996) ActaCrystallogr. D 52, 110-119).

The structure co-ordinates of the mutants of the N-domain of ACEprovided in this invention also facilitate the identification of relateddomains or enzymes analogous to the N-domain of ACE protein in function,structure or both, thereby further leading to novel therapeutic modesfor treating or preventing ACE related diseases.

The design of compounds that bind to or modulate the N-domain of ACEprotein or the C-domain of ACE protein according to the presentinvention generally involves consideration of two factors.

First, the compound must be capable of physically and structurallyassociating with the N-domain or the C-domain of ACE protein.Non-covalent molecular interactions important in the association of theN-domain of ACE with its substrate may include hydrogen bonding, van derWaals and hydrophobic interactions.

Second, the compound must be able to assume a conformation that allowsit to associate with the N-domain or C-domain of ACE protein. Althoughcertain portions of the compound may not directly participate in theassociation with the N-domain or C-domain of ACE, those portions maystill influence the overall conformation of the molecule. This may havea significant impact on potency of the compound. Such conformationalrequirements include the overall three-dimensional structure andorientation of the chemical ligand or compound in relation to all or aportion of a binding site of the N-domain or C-domain of ACE, or thespacing between functional groups of a compound comprising severalchemical entities that directly interact with the N-domain or C-domainof ACE.

The potential modulating or binding effect of a chemical compound on theN-domain or the C-domain of ACE may be analysed prior to its actualsynthesis and testing by the use of computer modelling techniques. Ifthe theoretical structure of the given compound suggests insufficientinteraction and association with the N-domain or the C-domain of ACE,then synthesis and testing of the compound may be obviated. However, ifcomputer modelling indicates a strong interaction, the molecule may besynthesised and tested for its ability to bind to the N-domain or theC-domain of ACE and modulate (e.g. inhibit) using the fluorescentsubstrate assay of Thornberry et al., (2000) Methods Enzymol. 322,100-110. In this manner, synthesis of inactive compounds may be avoided.

A modulating or other binding compound of the N-domain or the C-domainof ACE may be computationally evaluated and designed by means of aseries of steps in which chemical entities or test compounds arescreened and selected for their ability to associate with the N-domainor the C-domain of ACE.

A person skilled in the art may use one of several methods to screenchemical entities or test compounds for their ability to associate withthe N-domain or the C-domain of ACE and more particularly with theindividual binding sites of the N-domain or the C-domain of ACE. Thisprocess may begin by visual inspection of, for example, the active siteon the computer screen based on the N-domain or the C-domain of ACEco-ordinates of the present invention. Selected chemical entities ortest compounds may then be positioned in a variety of orientations, ordocked, with the N-domain or the C-domain of ACE. Docking may beaccomplished using software such as Quanta and Sybyl, followed by energyminimisation and molecular dynamics with standard molecular mechanicsforce fields—such as CHARMM and AMBER.

Specialised computer programs may also assist in the process ofselecting chemical entities or test compounds. These include but are notlimited to MCSS (Miranker and Karplus (1991) Proteins: Structure,Function and Genetics, 11, 29-34); GRID (Goodford (1985) J. Med. Chem.,28, 849-857) and AUTODOCK (Goodsell and Olsen (1990), Proteins:Structure. Function, and Genetics, 8, 195-202).

Once suitable chemical entities or test compounds have been selected,they may be assembled into a single compound capable of modulating thedomains of the ACE protein e.g. the N-domain and/or the C-domain of theACE protein. Assembly may proceed by visual inspection of therelationship of the chemical entities or test compounds in relation tothe structure co-ordinates of the N-domain of ACE protein. This may befollowed by manual model building using software—such as Quanta, Sybylor O [Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improvedmethods for building protein models in electron density maps and thelocation of errors in these models (1991) Acta Crystallogr. A 47,110-119].

Refinement of the model may be carried out using the program CNS[Brünger, A. T. et al., Crystallography & NMR System: A new softwaresuite for macromolecular structure determination. (1998) ActaCrystallogr. D 54, 905-921].

Various programs may be used by a skilled person to connect theindividual chemical entities or test compounds—such as 3D Databasesystems (Martin (1992) J. Med. Chem., 35, 2145-2154) and CAVEAT(Bartlett et al., (1989) Royal Chem. Soc. 78, 182-196).

Rather than build an inhibitor of the N-domain or the C-domain of ACEprotein one chemical ligand at a time, modelling of other N-domain orC-domain binding compounds may be designed as a whole or de novo usingeither an empty binding site or optionally including some portion(s) ofa known inhibitor(s). Such compounds may be designed using programs thatmay include but are not limited to LEGEND (Nishibata and Itai (1991)Tetrahedron, 47, 8985) and LUDI (Bohm (1992) J. Comp. Aid. Molec.Design, 6, 61-78).

Other molecular modelling techniques may also be employed in accordancewith this invention—such as those described by Cohen et al., J. Med.Chem., 33, 883-894 (1990); Navia and Murcko (1992) Current Opinions inStructural Biology, 2, 202-210 (1992).

Once a compound has been designed or selected by the above methods, theefficiency with which that compound may bind to the N-domain or theC-domain of ACE protein may be computationally evaluated. Specificcomputer software may be used to evaluate the efficiency of binding (eg.to evaluate compound deformation energy and electrostaticinteraction)—such as QUANTA/CHARMM (Accelrys Inc., USA) and InsightII/Discover (Biosym Technologies Inc., San Diego, Calif., USA). Theseprograms may be implemented, for instance, using a suitable workstation.Other hardware systems and software packages will be known to thosepersons skilled in the art.

Once a modulating compound has been selected or designed, as describedabove, substitutions may be made (eg. in atoms or side groups) toimprove or modify the binding properties. The substitutions may beconservative i.e. the replacement group may have approximately the samesize, shape, hydrophobicity and charge as the original group. Suchsubstituted chemical compounds may then be analysed for efficiency ofbinding to the N-domain or C-domain of ACE by the same computer methodsdescribed above.

Test compounds and modulators of the N-domain or C-domain of ACE proteinwhich are identified using the crystal and the methods of the presentinvention may be screened in assays. Screening can be, for example invitro, in cell culture, and/or in vivo. Biological screening assayspreferably centre on activity-based response models, binding assays(which measure how well a compound binds), and bacterial, yeast andanimal cell lines (which measure the biological effect of a compound ina cell). The assays can be automated for high capacity-high throughputscreening (HTS) in which large numbers of compounds can be tested toidentify compounds with the desired activity.

Current screening technologies are described in Handbook of DrugScreening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes. NewYork, N.Y., Marcel Dekker, (2001).

Disorders

These include, but are not limited to, treatment of high blood pressure;treatment of heart failure; prolonging survival of patients who have hada heart attack; preventing death by heart attack and stroke in patientswith vascular disease and in diabetics with other vascular risk factors;prolonging survival of patients with weak heart muscle; helping leakingheart valves; preserving kidney function in diabetics; and the treatmentof new indications (e.g. polycythemia). Special groups of patients mayalso be treated with N-domain or C-domain of ACE inhibitors, includingpatients with chronic pulmonary disease, patients with schleroderma,patients with atheroschlerosis, and patients with hyperuricemia.

It is also envisaged that the modulators identified by the methodsdescribed herein may be used to treat Alzheimer's disease and otherdegenerative diseases, conditions or disorders of the CNS.

Ace Constructs

The N-domain of ACE protein produced by a host recombinant cell may besecreted or may be contained intracellularly depending on the nucleotidesequence and/or the vector used.

The skilled person would understand that expression vectors containingthe N-domain of ACE protein encoding nucleotide sequence or a mutant,variant, homologue, derivative or fragment thereof, may be designed withsignal sequences which direct secretion of the N-domain of ACE proteincoding sequences across a particular prokaryotic or eukaryotic cellmembrane.

The N-domain encoding sequence may be fused (e.g. ligated) to nucleotidesequences encoding a polypeptide domain which will facilitatepurification of soluble proteins (Kroll D J et al., (1993) DNA Cell Biol12:441-53). Preferably, the polypeptide domain which facilitatespurification of soluble proteins is fused in frame with the N-domain ofthe ACE protein encoding sequence. Such purification facilitatingdomains include, but are not limited to, metal chelating peptides—suchas histidine-tryptophan modules that allow purification on immobilisedmetals (Porath J (1992) Protein Expr Purif 3, 263-281), protein Adomains that allow purification on immobilised immunoglobulin, and thedomain utilised in the FLAGS extension/affinity purification system(Immunex Corp, Seattle, Wash.). The inclusion of a cleavable linkersequence such as Factor XA or enterokinase (Invitrogen, San Diego,Calif.) between the purification domain and ACE is useful to facilitatepurification.

Preferably the N-domain of ACE protein sequence is fused with theglutathione synthetase (GS) signal.

Preferably, the N-domain of ACE protein is secreted in a soluble formhost cells and can be harvested from the growth medium.

Preferably, the N-domain of ACE construct is pEE14 N-domain whichencodes the N-domain of human tACE (SEQ ID NO: 2). This constructcomprises the inter-domain linker region.

Host Cell

As used herein, the term “host cell” refers to any cell that comprisesnucleotide sequences that are of use in the present invention, forexample, nucleotide sequences encoding the N-domain of ACE protein.

Host cells may be transformed or transfected with a nucleotide sequencecontained in a vector e.g. a cloning vector. Preferably, said nucleotidesequence is carried in a vector for the replication and/or expression ofthe nucleotide sequence. The cells will be chosen to be compatible withthe said vector and may for example be prokaryotic such as bacterial oreukaryotic such as fungal, yeast or plant cells.

The gram-negative bacterium E. coli is widely used as a host for cloningnucleotide sequences. This organism is also widely used for heterologousnucleotide sequence expression. However, large amounts of heterologousprotein tend to accumulate inside the cell. Subsequent purification ofthe desired protein from the bulk of E. coli intracellular proteins cansometimes be difficult.

In contrast to E. coli, bacteria from the genus Bacillus are verysuitable as heterologous hosts because of their capability to secreteproteins into the culture medium. Other bacteria suitable as hosts arethose from the genera Streptomyces and Pseudomonas.

Depending on the nature of the polynucleotide and/or the desirabilityfor further processing of the expressed protein, eukaryotic hostsincluding yeast or other fungi may be preferred. In general, yeast cellsare preferred over fungal cells because yeast cells are easier tomanipulate. However, some proteins are either poorly secreted from theyeast cell, or in some cases are not processed properly (e.g.hyperglycosylation). In these instances, a different fungal hostorganism should be selected.

Examples of expression hosts are fungi—such as Aspergillus species (suchas those described in EP-A-0184438 and EP-A-0284603) and Trichodermaspecies; bacteria—such as Bacillus species (such as those described inEP-A-0134048 and EP-A-0253455), Streptomyces species and Pseudomonasspecies; yeast—such as Kluyveromyces species (such as those described inEP-A-0096430 and EP-A-0301670) and Saccharomyces species; mammaliancells—such as CHO-K1 cells.

The use of host cells may provide for post-translational modifications(e.g. glycosylation) as may be needed to confer optimal biologicalactivity on recombinant expression products of the present invention.

Aspects of the present invention also relate to host cells comprisingthe N-domain of ACE protein constructs of the present invention. TheN-domain of ACE protein constructs may comprise a nucleotide sequencefor replication and expression of the sequence. The cells will be chosento be compatible with the vector and may for example be prokaryotic suchas bacterial or eukaryotic such as fungal, yeast or plant cells.

In a preferred embodiment, the host cells are mammalian cells—such asCHO-K1 cells. CHO-K1 cells expressing the N-domain of ACE protein may begrown and maintained in accordance with Yu et al. (1997).

Nucleotide Sequences

As used herein, the term “nucleotide sequence” refers to nucleotidesequences, oligonucleotide sequences, polynucleotide sequences andvariants, homologues, fragments and derivatives thereof (such asportions thereof) which comprise the nucleotide sequences encoding theN-domain of ACE protein, for example, the N-domain of testis ACE protein(SEQ ID NO: 2) or the N-domain (SEQ ID NO: 1) of somatic ACE protein.

The nucleotide sequence may be DNA or RNA of genomic or synthetic orrecombinant origin, which may be double-stranded or single-strandedwhether representing the sense or antisense strand or combinationsthereof.

Preferably, the term nucleotide sequence is prepared by use ofrecombinant DNA techniques (e.g. recombinant DNA). The nucleotidesequences may include within them synthetic or modified nucleotides. Anumber of different types of modification to oligonucleotides are knownin the art. These include methylphosphonate and phosphorothioatebackbones addition of acridine or polylysine chains at the 3′ and/or 5′ends of the molecule. For the purposes of the present invention, it isto be understood that the nucleotide sequences described herein may bemodified by any method available in the art.

It will be understood by a skilled person that numerous differentnucleotide sequences can encode the same protein as a result of thedegeneracy of the genetic code. In addition, it is to be understood thatskilled persons may, using routine techniques, make nucleotidesubstitutions that do not substantially affect the activity encoded bythe nucleotide sequence of the present invention to reflect the codonusage of any particular host organism in which the target is to beexpressed.

The terms “variant”, “homologue” or “derivative” in relation tonucleotide sequences include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence providing the resultantnucleotide sequence encodes a functional protein according to thepresent invention (or even a modulator of the N-domain of ACE proteinaccording to the present invention if said modulator comprises anucleotide sequence).

Amino Acid Sequences

As used herein, the term “amino acid sequence” is synonymous with theterm “polypeptide” and/or the term “protein”. In some instances, theterm “amino acid sequence” is synonymous with the term “peptide”.

Aspects of the present invention concern the use of amino acidsequences, which may be available in databases. These amino acidsequences may comprise N-domain of ACE proteins.

The amino acid sequence may be isolated from a suitable source, or itmay be made synthetically or it may be prepared by use of recombinantDNA techniques.

The terms “variant”, “homologue” or “derivative” in relation to aminoacid sequences include any substitution of, variation of, modificationof, replacement of, deletion of or addition of one (or more) amino acidfrom the functional protein according to the present invention (or evena modulator of the N-domain of ACE protein according to the presentinvention if said modulator comprises an amino acid sequence).

Preferably, the N-domain of ACE protein comprises SEQ ID NO: 1 or amutant, variant, homologue, derivative or a fragment thereof.

Purity

Preferably the protein solution used for crystallisation is at least95.5% pure. More preferably the protein solution used forcrystallisation is at least 97.5% pure. More preferably, the proteinsolution used for crystallisation is at least 99.0% pure. Mostpreferably, the protein solution used for crystallisation is at least99.5% pure.

Model

As used herein, the term “model” refers to a structural model such as athree dimensional (3D) structural model (or representation thereof)comprising the N-domain of ACE protein.

Test compounds can be modelled that bind spatially and preferentially tothe N-domain of ACE protein—such as to bind spatially and preferentiallyto the N-domain of ACE protein—for example, the active site of theN-domain of ACE protein.

Preferably, the crystal model comprising the N-domain of ACE is builtfrom all or a portion of the structural co-ordinates set forth in TableA (SEQ ID NOs: 5 and 6) or Table B (SEQ ID NOs: 7 and 8).

Mutant

As used herein, the term “mutant” refers to the N-domain of ACE proteincomprising any one or more changes in the wild-type ACE sequence shownas SEQ ID NO: 1.

The term “mutant” is not limited to any of the mutations describedherein which are reflected in amino acid substitutions of the amino acidresidues in the N-domain of ACE protein, but are not limited to, otherdeletions or insertions of nucleotides which may result in changes inthe amino acid residues in the amino acid sequence of the N-domain ofACE protein.

The present invention also enables the solving of the crystal structureof mutants of the N-domain of ACE protein. More particularly, by virtueof the present invention, the location of the active site of theN-domain of ACE protein based on its crystal structure permits theidentification of desirable sites for mutation. For example, one or moremutations may be directed to a particular site—such as the activesite—or combination of sites. Similarly, only a location on, at or nearthe inter-domain linker region may be replaced, resulting in an altereddomain co-operativity by changing the charge of one or more chargeunits, as compared to the wild-type N-domain. Alternatively, an aminoacid residue in the inter-domain linker region of the N-domain of ACEmay be chosen for replacement based on its hydrophilic or hydrophobiccharacteristics.

Such mutants may be characterised by any one of several differentproperties as compared with wild-type N-domain of ACE protein. Forexample, such mutants may have altered surface charge of one or morecharge units, or have an increased stability to subunit dissociation, oran altered substrate specificity in comparison with, or a higherspecific activity than, wild-type of the N-domain of ACE protein.

The mutants may be prepared in a number of ways that are known by aperson skilled in the art. For example, mutations may be introduced bymeans of oligonucleotide-directed mutagenesis or other conventionalmethods. Alternatively, mutants of the N-domain of ACE protein may begenerated by site specific replacement of a particular amino acid withan unnaturally occurring amino acid. This may be achieved by growing ahost organism capable of expressing either the wild-type or mutantpolypeptide on a growth medium depleted of one or more natural aminoacids but enriched in one or more corresponding unnaturally occurringamino acids.

The expression, activity (e.g. kinetic constants) and/or thecrystallisation properties of the mutants may be determined using themethods described herein.

Variants/Homologues/Derivatives/Fragments

The N-domain of ACE protein described herein is intended to include anypolypeptide, which has the activity of the naturally occurring N-domainand includes all vertebrate and mammalian forms. Such terms also includepolypeptides that differ from naturally occurring forms of the N-domainby having amino acid deletions, substitutions, and additions, but whichretain the activity of the N-domain of ACE protein.

The term “variant” is used to mean a naturally occurring polypeptide ornucleotide sequences which differs from a wild-type or a nativesequence.

The term “fragment” indicates that a polypeptide or nucleotide sequencecomprises a fraction of a wild-type or a native sequence. It maycomprise one or more large contiguous sections of sequence or aplurality of small sections. The sequence may also comprise otherelements of sequence, for example, it may be a fusion protein withanother protein. Preferably the sequence comprises at least 50%, morepreferably at least 65%, more preferably at least 80%, most preferablyat least 90% of the wild-type sequence.

The present invention also encompasses the use of variants, homologuesand derivatives of nucleotide and amino acid sequences. Here, the term“homologue” means an entity having a certain homology with amino acidsequences or nucleotide sequences. Here, the term “homology” can beequated with “identity”.

In the present context, a homologous sequence is taken to include anamino acid sequence which may be at least 75%, 85% or 90% identical,preferably at least 95% or 98% identical to the subject sequence.Although homology can also be considered in terms of similarity (i.e.amino acid residues having similar chemical properties/functions), it ispreferred here to express homology in terms of sequence identity.

Homologous sequence is taken to include a nucleotide sequence which maybe at least 75%, 85% or 90% identical, preferably at least 95% or 98%identical to the subject sequence.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

Homology when based on percentage (%) may be calculated over contiguoussequences, i.e. one sequence is aligned with the other sequence and eachamino acid in one sequence is directly compared with the correspondingamino acid in the other sequence, one residue at a time. This is usuallyreferred to “ungapped” alignment. Typically, such ungapped alignmentsare performed only over a relatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage the default gap penalty for amino acid sequences is −12 for agap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). However, for some applications, it is preferred to usethe GCG Bestfit program. A new tool, called BLAST 2 Sequences is alsoavailable for comparing protein and nucleotide sequence (see FEMSMicrobiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1):187-8)

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). For some applications, it ispreferred to use the public default values for the GCG package, or inthe case of other software, the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

By way of example, homologous sequences of the N-domain (SEQ ID NO: 1)of ACE protein include, but are not limited to human ACE_(S) somatic ACE(accession number: J04144), human ACE_(T) testis ACE (accession number:M26657) (SEQ ID NO: 2), human ACEH/ACE2 (accession numbers: AAF78220;BAB40370; AAF99721) (SEQ ID NO:3), chimp ACE_(T) (accession number:AF193487_2), rabbit ACE_(T) mature protein (accession number: P22968),rabbit ACE_(T) full pre-protein (accession number: P22968), mouseACE_(T) testis ACE (accession number: P22967), bovine Cdom ACE_(S)C-domain, rat Cdom ACE_(S) C-domain (derived from accession numberP47820; starting D616), human Ndom ACE_(S) N-domain (SEQ ID NO: 1)(derived from accession number P12821 (J04144)), chimp Ndom ACE_(S)N-domain (derived from accession number AF193487_1), rabbit Ndom ACE_(S)N-domain (derived from P12822), bovine Ndom (Bovine {Bos taurus} ACE_(S)N-domain), mouse Ndom ACE_(S) N-domain (derived from accession numberP09470), rat Ndom ACE_(S) N-domain (derived from accession numberP47820), chick ACE (partial ACE accession number Q10751), dros AnCE(derived from accession number Q10714), dros ACEr (derived fromaccession number X96913), buffalo fly ACE (derived from accession numberQ10715), and silkworm ACE (derived from accession number BAA97657), tickACE (derived from accession number U62809).

The sequences may also have deletions, insertions or substitutions ofamino acid residues, which produce a silent change and result in afunctionally equivalent substance. Deliberate amino acid substitutionsmay be made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the secondary binding activity of the substance isretained. For example, negatively charged amino acids include asparticacid and glutamic acid; positively charged amino acids include lysineand arginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine,glycine, alanine, asparagine, glutamine, serine, threonine,phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar -charged D E K R AROMATIC H F W Y

Homologous substitution (substitution and replacement are both usedherein to mean the interchange of an existing amino acid residue, withan alternative residue) may occur i.e. like-for-like substitution suchas basic for basic, acidic for acidic, polar for polar etc..Non-homologous substitution may also occur i.e. from one class ofresidue to another or alternatively involving the inclusion of unnaturalamino acids such as ornithine (hereinafter referred to as Z),diaminobutyric acid ornithine (hereinafter referred to as B), norleucineornithine (hereinafter referred to as O), pyriylalanine, thienylalanine,naphthylalanine and phenylglycine.

Replacements may also be made by unnatural amino acids include; alpha*and alpha-disubstituted* amino acids, N-alkyl amino acids*, lacticacid*, halide derivatives of natural amino acids such astrifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*,p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyricacid*, L-γ-amino butyric acid*, L-α-amino isobutyric acid*, L-ε-aminocaproic acid^(#), 7-amino heptanoic acid*, L-methionine sulfone^(#*),L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*,L-hydroxyproline^(#), L-thioproline*, methyl derivatives ofphenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe(4-amino)^(#), L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionicacid # and L-Phe (4-benzyl)*. The notation * has been utilised for thepurpose of the discussion above (relating to homologous ornon-homologous substitution), to indicate the hydrophobic nature of thederivative whereas # has been utilised to indicate the hydrophilicnature of the derivative, #* indicates amphipathic characteristics.

The term “derivative” or “derivatised” as used herein includes chemicalmodification of a ligand—such as test compound or a modulator of theN-domain of ACE protein. Illustrative of such chemical modificationswould be replacement of hydrogen by a halo group, an alkyl group, anacyl group or an amino group.

Variant amino acid sequences may include suitable spacer groups that maybe inserted between any two amino acid residues of the sequenceincluding alkyl groups such as methyl, ethyl or propyl groups inaddition to amino acid spacers such as glycine or (β-alanine residues. Afurther form of variation, involves the presence of one or more aminoacid residues in peptoid form, will be well understood by those skilledin the art. For the avoidance of doubt, “the peptoid form” is used torefer to variant amino acid residues wherein the α-carbon substituentgroup is on the residue's nitrogen atom rather than the α-carbon.Processes for preparing peptides in the peptoid form are known in theart, for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 andHorwell D C, Trends Biotechnol. (1995) 13(4), 132-134.

Test Compound

As used herein, the term “test compound” includes, but is not limitedto, a compound which may be obtainable from or produced by any suitablesource, whether natural or not.

The test compound may be designed or obtained from a small moleculelibrary of compounds, which may comprise peptides, as well as othercompounds, such as small organic molecules and particularly new leadcompounds. By way of example, the test compound may be a naturalsubstance, a biological macromolecule, or an extract made frombiological materials—such as bacteria, fungi, or animal (particularlymammalian) cells or tissues, an organic or an inorganic molecule, asynthetic test compound, a semi-synthetic test compound, a structural orfunctional mimetic, a peptide, a peptidomimetics, a derivatised testcompound, a peptide cleaved from a whole protein, or a peptidesynthesised synthetically (such as, by way of example, either using apeptide synthesiser or by recombinant techniques or combinationsthereof, a recombinant test compound, a natural or a non-natural testcompound, a fusion protein or equivalent thereof and mutants,derivatives or combinations thereof. The test compound may even be acompound that is a modulator of the N-domain or C-domain of ACEprotein—such as a known inhibitor of the N-domain or C-domain of ACEprotein—that has been modified in some way e.g. by recombinant DNAtechniques or chemical synthesis techniques.

Typically, the test compound will be prepared by recombinant DNAtechniques and/or chemical synthesis techniques.

Once a test compound capable of interacting with the N-domain orC-domain of ACE protein has been identified, further steps may becarried out to select and/or to modify the test compounds and/or tomodify existing compounds, such that they are able to modulate theN-domain or C-domain of ACE protein.

The present invention also relates to a test compound which may be adomain specific compound such as a domain specific inhibitor capable ofmodulating the activity of the ACE protein in a domain specific manner.That is the compound is capable of modulating the N-domain and/orC-domain of ACE protein.

Modulating the Activity of Ace

As herein, the term “modulating” refers to preventing, suppressing,inhibiting, alleviating, restoring, elevating, increasing or otherwiseaffecting the N-domain or C-domain of ACE protein.

The term “modulator of N-domain or C-domain of ACE” may refer to asingle ligand or a combination of ligands.

The modulator of the N-domain or C-domain of ACE protein may be anantagonist or an agonist of the N-domain or the C-domain of ACE.

As used herein, the term “agonist” means any ligand, which is capable ofinteracting (e.g. binding) with N-domain or C-domain of ACE protein andwhich is capable of increasing a proportion of the N-domain or C-domainof ACE that is in an active form, resulting in an increased biologicalresponse.

As used herein, the term “antagonist” means any ligand, which is capableof interacting (e.g. binding) with N-domain or C-domain of ACE proteinand which is capable of decreasing (eg. inhibiting) a proportion of theN-domain or C-domain of ACE that is in an active form, resulting in adecreased biological response.

Preferably, the modulators of the N-domain or C-domain of ACE protein ofthe present invention are antagonists of the N-domain or C-domain of ACEprotein.

The modulator may be an organic compound or other chemical. Themodulator may be a compound, which is obtainable from or produced by anysuitable source, whether natural or artificial. The modulator may be anamino acid molecule, a polypeptide, or a chemical derivative thereof, ora combination thereof. The modulator may even be a polynucleotidemolecule—which may be a sense or an anti-sense molecule. The modulatormay even be an antibody.

The modulator of the N-domain or C-domain of ACE protein may be designedor obtained from a small molecule library of compounds, which maycomprise peptides, as well as other compounds or small organicmolecules.

By way of example, the modulator of the N-domain or C-domain of ACEprotein may be a natural substance, a biological macromolecule, or anextract made from biological materials such as bacteria, fungi, oranimal (particularly mammalian) cells or tissues, an organic or aninorganic molecule, a synthetic agent, a semi-synthetic agent, astructural or functional mimetic, a peptide, a peptidomimetic, aderivatised agent, a peptide cleaved from a whole protein, or a peptidesynthesised synthetically (such as, by way of example, either using apeptide synthesiser or by recombinant techniques or combinationsthereof, a recombinant agent, an antibody, a natural or a non-naturalagent, a fusion protein or equivalent thereof and mutants, derivativesor combinations thereof).

Typically, the modulator of the N-domain or C-domain of ACE protein willbe an organic compound. Typically, the organic compounds will comprisetwo or more hydrocarbyl groups. Here, the term “hydrocarbyl group” meansa group comprising at least C and H and may optionally comprise one ormore other suitable substituents. Examples of such substituents mayinclude halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. Inaddition to the possibility of the substituents being a cyclic group, acombination of substituents may form a cyclic group. If the hydrocarbylgroup comprises more than one C then those carbons need not necessarilybe linked to each other. For example, at least two of the carbons may belinked via a suitable element or group. Thus, the hydrocarbyl group maycontain hetero atoms. Suitable hetero atoms will be apparent to thoseskilled in the art and include, for instance, sulphur, nitrogen andoxygen. For some applications, preferably the modulator of the N-domainor C-domain of ACE protein comprises at least one cyclic group. Thecyclic group may be a polycyclic group, such as a non-fused polycyclicgroup. For some applications, the modulator of the N-domain or C-domainof ACE protein comprises at least the one of said cyclic groups linkedto another hydrocarbyl group.

The modulator of the N-domain or C-domain of ACE protein may containhalo groups, for example, fluoro, chloro, bromo or iodo groups.

The modulator of the N-domain or C-domain of ACE protein may contain oneor more of alkyl, alkoxy, alkenyl, alkylene and alkenylene groups—whichmay be unbranched- or branched-chain.

The modulator of the N-domain or C-domain of ACE protein may be in theform of a pharmaceutically acceptable salt—such as an acid addition saltor a base salt—or a solvate thereof, including a hydrate thereof. For areview on suitable salts see Berge et al., (1977) J. Pharm. Sci. 66,1-19.

The modulator of the N-domain or C-domain of ACE protein may be astructurally novel modulator.

The modulators of the N-domain or C-domain of ACE protein may beanalogues to other known modulators—such as known inhibitors of theN-domain of ACE protein (for example, snake venom, peptides produced byenzymatic hydrolysis of casein or fish meat protein, or Benazepril,Captopril, Cilazapril, Enalapril, Fosinopril, Lisinopril, Moexipril,Perindopril, Quinapril, Ramipril, Trandolapril and Enalaprilat).

Preferably the inhibitor of the N-domain or C-domain of ACE protein isLisinopril (N2-[(S)-1-carboxy-3-phenylpropyl]-L-lysyl-L-proline; alsoknown as Prinivil or Zestril).

Preferably, the N-domain or C-domain of ACE protein modulators haveimproved properties over those previously available, for example, fewerside effects—such as cough (e.g. dry, persistent); fever and chills;hoarseness; swelling of face, mouth, hands, or feet; trouble inswallowing or breathing; itching of skin; yellow eyes or skin;dizziness, light-headedness, or fainting; skin rash, with or withoutitching; fever, or joint pain; abdominal pain, abdominal distention;nausea, or vomiting; chest pain, confusion; irregular heartbeat;nervousness; numbness or tingling in hands, feet, or lips; weakness orheaviness of legs; headache, diarrhoea; loss of taste; nausea; unusualtiredness and angioedema.

The modulator of the N-domain or C-domain of ACE protein may be amimetic.

The modulator of the N-domain or C-domain of ACE protein may also bechemically modified.

The modulator of the N-domain or C-domain of ACE protein may be capableof displaying other therapeutic properties.

The modulator of the N-domain or C-domain of ACE protein may be used incombination with one or more other pharmaceutically active agents.

If combinations of active agents are administered, then these may beadministered simultaneously, separately or sequentially.

Mimetic

As used herein, the term “mimetic” relates to any chemical including,but not limited to, peptide, polypeptide, antibody or other organicchemical with the same qualitative activity or effect as a knowncompound. That is, the mimetic is a functional equivalent of a knowncompound.

Stereo and Geometric Isomers

Modulators of the N-domain or C-domain of ACE protein may exist asstereoisomers and/or geometric isomers—e.g. they may possess one or moreasymmetric and/or geometric centres and so may exist in two or morestereoisomeric and/or geometric forms. The present inventioncontemplates the use of all of the individual stereoisomers andgeometric isomers, and mixtures thereof.

Pharmaceutical Salt

Modulators of the N-domain or C-domain of ACE protein may beadministered in the form of a pharmaceutically acceptable salt.

Pharmaceutically-acceptable salts are well known to those skilled in theart, and for example include those mentioned by Berge et al., (1977) J.Pharm. Sci., 66, 1-19. Suitable acid addition salts are formed fromacids which form non-toxic salts and include the hydrochloride,hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate,hydrogenphosphate, acetate, trifluoroacetate, gluconate, lactate,salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate,gluconate, formate, benzoate, methanesulphonate, ethanesulphonate,benzenesulphonate and p-toluenesulphonate salts.

When one or more acidic moieties are present, suitable pharmaceuticallyacceptable base addition salts can be formed from bases which formnon-toxic salts and include the aluminium, calcium, lithium, magnesium,potassium, sodium, zinc, and pharmaceutically-active amines such asdiethanolamine, salts.

A pharmaceutically acceptable salt of a modulator of the N-domain orC-domain of ACE protein may be readily prepared by mixing togethersolutions of the modulator and the desired acid or base, as appropriate.The salt may precipitate from solution and be collected by filtration ormay be recovered by evaporation of the solvent.

The modulator of the N-domain or C-domain of ACE protein may exist inpolymorphic form.

The modulator of the N-domain or C-domain of ACE protein may contain oneor more asymmetric carbon atoms and therefore exists in two or morestereoisomeric forms. Where a modulator of the N-domain or C-domain ofACE protein contains an alkenyl or alkenylene group, cis (E) and trans(Z) isomerism may also occur. The present invention includes theindividual stereoisomers of the modulator of the N-domain or C-domain ofACE protein and, where appropriate, the individual tautomeric formsthereof, together with mixtures thereof.

Separation of diastereoisomers or cis and trans isomers may be achievedby conventional techniques, e.g. by fractional crystallisation,chromatography or H.P.L.C. of a stereoisomeric mixture of the modulatorof the N-domain or C-domain of ACE protein or a suitable salt orderivative thereof. An individual enantiomer of the modulator of theN-domain or C-domain of ACE may also be prepared from a correspondingoptically pure intermediate or by resolution, such as by H.P.L.C. of thecorresponding racemate using a suitable chiral support or by fractionalcrystallisation of the diastereoisomeric salts formed by reaction of thecorresponding racemate with a suitable optically active acid or base, asappropriate.

The modulator of the N-domain or C-domain of ACE may also include allsuitable isotopic variations of the modulator or a pharmaceuticallyacceptable salt thereof. An isotopic variation of an modulator of theN-domain or C-domain of ACE or a pharmaceutically acceptable saltthereof is defined as one in which at least one atom is replaced by anatom having the same atomic number but an atomic mass different from theatomic mass usually found in nature. Examples of isotopes that can beincorporated into the modulator of ACE and pharmaceutically acceptablesalts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorus, sulphur, fluorine and chlorine such as ²H, ³H, ¹³C, ¹⁴C,¹⁵N, ¹⁷O ¹⁸O ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶Cl, respectively. Certain isotopicvariations of the modulator the N-domain or C-domain of ACE andpharmaceutically acceptable salts thereof, for example, those in which aradioactive isotope such as ³H or ¹⁴C is incorporated, are useful indrug and/or substrate tissue distribution studies. Tritiated, i.e., ³H,and carbon-14, i.e., ¹⁴C, isotopes are particularly preferred for theirease of preparation and detectability. Further, substitution withisotopes such as deuterium, i.e., ²H, may afford certain therapeuticadvantages resulting from greater metabolic stability, for example,increased in vivo half-life or reduced dosage requirements and hence maybe preferred in some circumstances. Isotopic variations of the modulatorof the N-domain or C-domain of ACE and pharmaceutically acceptable saltsthereof of this invention can generally be prepared by conventionalprocedures using appropriate isotopic variations of suitable reagents.

It will be appreciated by those skilled in the art that the agent may bederived from a prodrug. Examples of prodrugs include entities that havecertain protected group(s) and which may not possess pharmacologicalactivity as such, but may, in certain instances, be administered (suchas orally or parenterally) and thereafter metabolised in the body toform the modulator of the N-domain or C-domain of ACE which ispharmacologically active.

It will be further appreciated that certain moieties known as“pro-moieties”, for example as described in “Design of Prodrugs” by H.Bundgaard, Elsevier, 1985 (the disclosured of which is herebyincorporated by reference), may be placed on appropriate functionalitiesof the modulator of the N-domain or C-domain of ACE protein. Suchprodrugs are also contemplated within the scope of the invention.

Pharmaceutically Active Salt

The modulator of the N-domain or C-domain of ACE protein may beadministered as a pharmaceutically acceptable salt. Typically, apharmaceutically acceptable salt may be readily prepared by using adesired acid or base, as appropriate. The salt may precipitate fromsolution and be collected by filtration or may be recovered byevaporation of the solvent.

Chemical Synthesis Methods

The modulator of the N-domain or C-domain of ACE protein of the presentinvention may be prepared by chemical synthesis techniques.

It will be apparent to those skilled in the art that sensitivefunctional groups may need to be protected and deprotected duringsynthesis of a compound of the invention. This may be achieved byconventional techniques, for example as described in “Protective Groupsin Organic Synthesis” by T W Greene and P G M Wuts, John Wiley and SonsInc. (1991), and by P. J. Kocienski, in “Protecting Groups”, GeorgThieme Verlag (1994).

It is possible during some of the reactions that any stereocentrespresent could, under certain conditions, be racemised, for example if abase is used in a reaction with a substrate having an having an opticalcentre comprising a base-sensitive group. This is possible during e.g. aguanylation step. It should be possible to circumvent potential problemssuch as this by choice of reaction sequence, conditions, reagents,protection/deprotection regimes, etc. as is well-known in the art.

The compounds and salts may be separated and purified by conventionalmethods.

Separation of diastereomers may be achieved by conventional techniques,e.g. by fractional crystallisation, chromatography or H.P.L.C. of astereoisomeric mixture of a compound of formula (I) or a suitable saltor derivative thereof. An individual enantiomer of a compound of formula(I) may also be prepared from a corresponding optically pureintermediate or by resolution, such as by H.P.L.C. of the correspondingracemate using a suitable chiral support or by fractionalcrystallisation of the diastereomeric salts formed by reaction of thecorresponding racemate with a suitably optically active acid or base.

The N-domain or C-domain of ACE protein, modulators of the N-domain orC-domain of ACE protein or variants, homologues, derivatives, fragmentsor mimetics thereof may be produced using chemical methods to synthesisethe N-domain or C-domain of ACE protein or the modulator of the N-domainor C-domain of ACE protein in whole or in part. For example, theN-domain or C-domain peptide or a modulator of the N-domain or C-domainof ACE protein that is a peptide can be synthesised by solid phasetechniques, cleaved from the resin, and purified by preparative highperformance liquid chromatography (e.g., Creighton (1983) ProteinsStructures And Molecular Principles, WH Freeman and Co, New York N.Y.).The composition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure;Creighton, supra).

Synthesis of peptides (or variants, homologues, derivatives, fragmentsor mimetics thereof) may be performed using various solid-phasetechniques (Roberge J Y et al (1995) Science 269: 202-204) and automatedsynthesis may be achieved, for example, using the ABI 43 1 A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer. Additionally, the amino acid sequences comprisingthe modulator of the N-domain or C-domain of ACE protein, may be alteredduring direct synthesis and/or combined using chemical methods with asequence from other subunits, or any part thereof, to produce a variantmodulator of the N-domain or C-domain of ACE protein.

Chemical Modification

The modulator of the N-domain or C-domain of ACE protein may be achemically modified modulator.

The chemical modification of a modulator of the N-domain or C-domain ofACE protein may either enhance or reduce interactions between themodulator of the N-domain or C-domain of ACE protein and the target—suchas hydrogen bonding interactions, charge interactions, hydrophobicinteractions, van der Waals interactions or dipole interactions.

In one aspect, the modulator of the N-domain or C-domain of ACE proteinmay act as a model (for example, a template) for the development ofother compounds.

Pharmaceutical Compositions

The components may be administered alone but will generally beadministered as a pharmaceutical composition—e.g. when the componentsare in a mixture with a suitable pharmaceutical excipient, diluent orcarrier selected with regard to the intended route of administration andstandard pharmaceutical practice.

For example, the components can be administered in the form of tablets,capsules, ovules, elixirs, solutions or suspensions, which may containflavouring or colouring agents, for immediate-, delayed-, modified-,sustained-, pulsed- or controlled-release applications.

If the pharmaceutical is a tablet, then the tablet may containexcipients such as microcrystalline cellulose, lactose, sodium citrate,calcium carbonate, dibasic calcium phosphate and glycine, disintegrantssuch as starch (preferably corn, potato or tapioca starch), sodiumstarch glycollate, croscarmellose sodium and certain complex silicates,and granulation binders—such as polyvinylpyrrolidone,hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),sucrose, gelatin and acacia. Additionally, lubricating agents—such asmagnesium stearate, stearic acid, glyceryl behenate and talc may beincluded.

Solid compositions of a similar type may also be employed as fillers ingelatin capsules. Preferred excipients in this regard include lactose,starch, a cellulose, milk sugar or high molecular weight polyethyleneglycols. For aqueous suspensions and/or elixirs, the modulator of theN-domain or C-domain of ACE protein may be combined with varioussweetening or flavouring agents, colouring matter or dyes, withemulsifying and/or suspending agents and with diluents such as water,ethanol, propylene glycol and glycerin, and combinations thereof.

The routes for administration (delivery) may include, but are notlimited to, one or more of oral (e.g. as a tablet, capsule, or as aningestable solution), topical, mucosal (e.g. as a nasal spray or aerosolfor inhalation), nasal, parenteral (e.g. by an injectable form),gastrointestinal, intraspinal, intraperitoneal, intramuscular,intravenous, intraventricular, intrauterine, intraocular, intradermal,intracranial, intratracheal, intravaginal, intracerebroventricular,intracerebral, subcutaneous, ophthalmic (including intravitreal orintracameral), transdermal, rectal, buccal, vaginal, epidural,sublingual.

Pharmaceutical compositions of the present invention may comprise atherapeutically effective amount of the N-domain of ACE protein, one ormore modulators of the N-domain of ACE protein, one or more modulatorsof the C-domain of ACE protein or combinations thereof.

The pharmaceutical compositions may be for human or animal usage inhuman and veterinary medicine and will typically comprise any one ormore of a pharmaceutically acceptable diluent, carrier, or excipient.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as—or in addition to—the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Preservatives, stabilisers, dyes and even flavouring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

There may be different composition/formulation requirements dependent onthe different delivery systems. By way of example, the pharmaceuticalcomposition of the present invention may be formulated to beadministered using a mini-pump or by a mucosal route, for example, as anasal spray or aerosol for inhalation or ingestable solution, orparenterally in which the composition is formulated by an injectableform, for delivery, by, for example, an intravenous, intramuscular orsubcutaneous route. Alternatively, the formulation may be designed to beadministered by a number of routes.

If the modulator of the N-domain or C-domain of ACE protein is to beadministered mucosally through the gastrointestinal mucosa, it should beable to remain stable during transit though the gastrointestinal tract;for example, it should be resistant to proteolytic degradation, stableat acid pH and resistant to the detergent effects of bile.

Where appropriate, the pharmaceutical compositions may be administeredby inhalation, in the form of a suppository or pessary, topically in theform of a lotion, solution, cream, ointment or dusting powder, by use ofa skin patch, orally in the form of tablets containing excipients suchas starch or lactose, or in capsules or ovules either alone or inadmixture with excipients, or in the form of elixirs, solutions orsuspensions containing flavouring or colouring agents, or thepharmaceutical compositions can be injected parenterally, for exampleintravenously, intramuscularly or subcutaneously. For parenteraladministration, the compositions may be best used in the form of asterile aqueous solution which may contain other substances, for exampleenough salts or monosaccharides to make the solution isotonic withblood. For buccal or sublingual administration the compositions may beadministered in the form of tablets or lozenges which can be formulatedin a conventional manner.

The modulators of the N-domain or C-domain of ACE protein may be used incombination with a cyclodextrin. Cyclodextrin molecules are known toform inclusion and non-inclusion complexes with drug molecules.Formation of a drug-cyclodextrin complex may modify the solubility,dissolution rate, bioavailability and/or stability property of a drugmolecule. Drug-cyclodextrin complexes are generally useful for mostdosage forms and administration routes. As an alternative to directcomplexation with the drug the cyclodextrin may be used as an auxiliaryadditive, e.g. as a carrier, diluent or solubiliser. Alpha-, beta- andgamma-cyclodextrins are most commonly used and suitable examples aredescribed in WO-A-91/11172, WO-A-94/02518 and WO-A-98/55148.

If the modulator of the N-domain or C-domain of ACE is a protein, thensaid protein modulator may be prepared in situ in the subject beingtreated. In this respect, nucleotide sequences encoding said protein maybe delivered by use of non-viral techniques (e.g. by use of liposomes)and/or viral techniques (e.g. by use of retroviral vectors) such thatthe said protein is expressed from said nucleotide sequence.

Dose Levels

Typically, a physician will determine the actual dosage which will bemost suitable for an individual subject. The specific dose level andfrequency of dosage for any particular patient may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, general health, sex, diet, mode and timeof administration, rate of excretion, drug combination, the severity ofthe particular condition, and the individual undergoing therapy.

The modulators of the N-domain of ACE protein or the C-domain of ACEprotein may be administered separately, concomitantly or sequentially.

Formulation

The component(s) may be formulated into a pharmaceutical composition,such as by mixing with one or more of a suitable carrier, diluent orexcipient, by using techniques that are known in the art.

Vector

Aspects of the present invention relate to a vector comprising anucleotide sequence—such as a nucleotide sequence encoding the N-domainor the C-domain of ACE protein or a modulator of the N-domain or theC-domain of ACE protein—administered to a subject.

Preferably, the N-domain or the C-domain of ACE and/or the modulator areprepared and/or delivered using a genetic vector.

It is well known in the art, that a vector is a tool that allows orfacilitates the transfer of information from one environment to another.In accordance with the present invention, and by way of example, somevectors used in recombinant DNA techniques allow entities, such as asegment of DNA (such as a heterologous DNA segment, such as aheterologous cDNA segment), to be transferred into a host and/or atarget cell for the purpose of replicating the vectors comprisingnucleotide sequences and/or expressing the proteins encoded by thenucleotide sequences. Examples of vectors used in recombinant DNAtechniques include, but are not limited to, plasmids, chromosomes,artificial chromosomes or viruses.

The term “vector” includes expression vectors such as transfectionvectors, transduction vectors or transformation vectors.

The term “expression vector” means a construct capable of in vivo or invitrolex vivo expression.

The terms “transfection vectors”, “transduction vectors” or“transformation vectors” describe different constructs capable of beingtransferred from one organism to another of from one species to another.

Regulatory Sequences

In some applications, nucleotide sequences are operably linked to aregulatory sequence which is capable of providing for the expression ofthe nucleotide sequence, such as by a chosen host cell. By way ofexample, a vector comprising the N-domain or the C-domain of ACEnucleotide sequence is operably linked to such a regulatory sequencei.e. the vector is an expression vector.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A regulatory sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences.

The term “regulatory sequences” includes promoters and enhancers andother expression regulation signals.

The term “promoter” is used in the normal sense of the art, e.g. an RNApolymerase binding site.

Enhanced expression of a nucleotide sequence, for example, a nucleotidesequence encoding the N-domain or the C-domain of ACE protein—may alsobe achieved by the selection of heterologous regulatory regions, e.g.promoter, secretion leader and terminator regions, which serve toincrease expression and, if desired, secretion levels of the protein ofinterest from the chosen expression host and/or to provide for theinducible control of the expression of the N-domain or the C-domain ofACE protein. In eukaryotes, polyadenylation sequences may be operablyconnected to the C-terminus of the nucleotide sequence of the N-domainor the C-domain of ACE protein.

Preferably, the N-domain or the C-domain of ACE protein nucleotidesequence is operably linked to at least a promoter.

Aside from the promoter native to the nucleotide sequence describedherein, other promoters may be used to direct its expression. Thepromoter may be selected for its efficiency in directing the expressionof the N-domain or the C-domain of ACE nucleotide sequence in thedesired expression host.

In another embodiment, a constitutive promoter may be selected to directthe expression of the N-domain or the C-domain of ACE nucleotidesequence of the present invention. Such an expression construct mayprovide additional advantages since it circumvents the need to culturethe expression hosts on a medium containing an inducing substrate.

Hybrid promoters may also be used to improve inducible regulation of theexpression construct.

The promoter can additionally include features to ensure or to increaseexpression in a suitable host. For example, the features can beconserved regions such as a Pribnow Box or a TATA box. The promoter mayeven contain other sequences to affect (such as to maintain, enhance,decrease) the levels of expression of the N-domain or C-domain of ACEnucleotide sequence. For example, suitable other sequences include theSh1-intron or an ADH intron. Other sequences include inducibleelements—such as temperature, chemical, light or stress inducibleelements. Also, suitable elements to enhance transcription ortranslation may be present.

Expression Vector

Preferably, nucleotide sequences—such as nucleotide sequences encodingthe N-domain or C-domain of ACE or modulators of the N-domain orC-domain of ACE—are inserted into a vector that is operably linked to acontrol sequence that is capable of providing for the expression of thecoding sequence by the host cell.

Nucleotide sequences produced by a host recombinant cell may be secretedor may be contained intracellularly depending on the sequence and/or thevector used. As will be understood by those of skill in the art,expression vectors can be designed with signal sequences, which directsecretion of the nucleotide sequence through a particular prokaryotic oreukaryotic cell membrane.

The expression vector may be pEE14 N-domain (SEQ ID NO: 4) which encodesthe N-domain of human ACE and is truncated in the interdomain linkerregion.

Preferably, the expression vectors are stably expressed in CHO-K1 cellsas described previously (Ehlers et al., (1996) Biochemistry 35,9549-9559).

Fusion Proteins

The N-domain or C-domain of ACE protein or a modulator of the N-domainor C-domain of ACE protein may be expressed as a fusion protein to aidextraction and purification and/or delivery of the modulator of theN-domain or C-domain of ACE or the N-domain or C-domain of ACE proteinto an individual and/or to facilitate the development of a screen formodulators of the N-domain or C-domain of ACE protein.

Examples of fusion protein partners include glutathione-S-transferase(GST), 6xHis, GAL4 (DNA binding and/or transcriptional activationdomains) and β-galactosidase.

It may also be convenient to include a proteolytic cleavage site betweenthe fusion protein partner and the protein sequence of interest to allowremoval of fusion protein sequences. Preferably, the fusion protein willnot hinder the activity of the protein of interest.

The fusion protein may comprise an antigen or an antigenic determinantfused to the substance of the present invention. In this embodiment, thefusion protein may be a non-naturally occurring fusion proteincomprising a substance, which may act as an adjuvant in the sense ofproviding a generalised stimulation of the immune system.

The antigen or antigenic determinant may be attached to either the aminoor carboxy terminus of the substance.

Organism

The term “organism” in relation to the present invention includes anyorganism that could comprise the N-domain or C-domain of ACE proteinand/or modulators of the N-domain or C-domain of ACE protein. Examplesof organisms may include mammals, fungi, yeast, plants or bacteria.

Preferably, the organism is as vertebrate or a mammal. More preferably,the organism is a human.

Transformation

As indicated earlier, the host organism can be a prokaryotic or aeukaryotic organism. Examples of suitable prokaryotic hosts include E.coli and Bacillus subtilis. Teachings on the transformation ofprokaryotic hosts are well documented in the art, for example seeSambrook et al., (Molecular Cloning: A Laboratory Manual, 2nd edition,1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., CurrentProtocols in Molecular Biology (1995), John Wiley & Sons, Inc. Examplesof suitable eukaryotic hosts include mammalian cells.

If a prokaryotic host is used then the nucleotide sequence—such as theN-domain of ACE nucleotide sequence—may need to be suitably modifiedbefore transformation—such as by removal of introns.

Thus, the present invention also relates to the transformation of a hostcell with a nucleotide sequence—such as those coding for the N-domain orC-domain of ACE protein or a modulator of the N-domain or C-domain ofACE protein. Host cells transformed with the nucleotide sequence may becultured under conditions suitable for the expression and recovery ofthe encoded protein from cell culture. The protein produced by arecombinant cell may be secreted or may be contained intracellularlydepending on the sequence and/or the vector used. As will be understoodby those of skill in the art, expression vectors containing codingsequences can be designed with signal sequences which direct secretionof the coding sequences through a particular prokaryotic or eukaryoticcell membrane. Other recombinant constructions may join the codingsequence to nucleotide sequence encoding a polypeptide domain, whichwill facilitate purification of soluble proteins (Kroll D J et al.,(1993) DNA Cell Biol 12:441-53) e.g. 6-His or Glutathione-S-transferase.

Transfection

Vectors comprising for example, the nucleotide sequence coding for theN-domain or C-domain of ACE protein, may be introduced into host cells,for example, mammalian cells, using a variety of methods.

Typical transfection methods include electroporation, DNA biolistics,lipid-mediated transfection, compacted DNA-mediated transfection,liposomes, immunoliposomes, lipofectin, cationic agent-mediated,cationic facial amphiphiles (CFAs) (Nature Biotech. (1996) 14, 556),multivalent cations such as spermine, cationic lipids or polylysine, 1,2,-bis (oleoyloxy)-3-(trimethylammonio) propane (DOTAP)-cholesterolcomplexes (Wolff and Trubetskoy (1998) Nature Biotech. 16, 421) andcombinations thereof.

Uptake of nucleic acid constructs by mammalian cells is enhanced byseveral known transfection techniques for example those including theuse of transfection agents. Example of these agents include cationicagents (for example calcium phosphate and DEAE-dextran) and lipofectants(for example Lipofectam™ and Transfectam™). Typically, nucleic acidconstructs are mixed with the transfection agent to produce acomposition.

Such methods are described in many standard laboratory manuals—such asSambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

General Recombinant DNA Methodology Techniques

The present invention employs, unless otherwise indicated, conventionaltechniques of chemistry, molecular biology, microbiology, recombinantDNA and immunology, which are within the capabilities of a person ofordinary skill in the art. Such techniques are explained in theliterature. See, for example, J. Sambrook, E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition,Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al.,(1995 and periodic supplements; Current Protocols in Molecular Biology,ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J.Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: EssentialTechniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990,In Situ Hybridization: Principles and Practice; Oxford University Press;M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A PracticalApproach, Irl Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992,Methods of Enzymology: DNA Structure Part A: Synthesis and PhysicalAnalysis of DNA Methods in Enzymology, Academic Press. Each of thesegeneral texts is herein incorporated by reference.

DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. (A) Overall structure of the N-domain (SEQ ID NO: 1) with thezinc ion in green, the chloride ion in red and the oligosaccharides inpink. (B) Superposition of the N-domain (SEQ ID NO: 1) (turquoise) andtACE (pink) (SEQ ID NO: 2) with the zinc ion in green. (C) Backbonecoloured according to thermal vibration (B factor) with blue for thelowest B factors and red for the highest. (D) Surface diagram showingthe lid (dark blue), N-linked glycans (green) and the protruding surfacepatch (pink) comprising the linker and the flexible loop.

FIG. 2. (A) Model of the possible domain orientation of ACE. TheN-terminus and C-terminal linker of two N-domains (SEQ ID NO: 1) (grey)were overlapped and tACE (pink) (SEQ ID NO: 2) superimposed. Sugarsmoieties visible in the N-domain (SEQ ID NO: 1) and tACE (SEQ ID NO: 2)structures are shown in green, and N-glycosylation sequons where nosugar was observed have been marked by a hypothetical sugar in yellow.The zinc ion in each domain is in green. (B) Close up of the N domainlinker and flexible loop (grey). (C) Pink domain lid showing theresidues that might be involved in the inter-domain interaction. Chargedresidues on the C-terminal lid are shown in red (negative) and blue(positive) with the sugar linked asparagine shown in green. Charged andsugar linked residues are marked by the same colours on the N-domain(SEQ ID NO: 1) ACE, but their side chains are not visible in thestructure.

FIG. 3. (A) Close up of the active site of the N-domain (SEQ ID NO: 1)ACE (blue with white residues) and tACE (pink with purple residues) (SEQID NO: 2) with the zinc ion in green and the conserved chloride ion inred. Lisinopril is shown in white/purple and the differing residuesbetween the N- and C-domains shown in ball and stick at the top of thepicture. (B) Close up of the minimally glycosylated N-domain (SEQ IDNO: 1) active site (blue) with a model of RXP407 (yellow) overlappingthe water molecule (grey) adjacent to Tyr 369. Residues differingbetween the N- and C-domains in the S₂ subsite are also shown in balland stick along with the conserved residues of the S₁ subsite thatinteract with lisinopril. Residues conserved between the N- (SEQ IDNO: 1) and C-domains are shown in white, N-domain (SEQ ID NO: 1)residues shown in blue and C-domain residues shown in pink. The zinc ionis shown in green.

FIG. 4. Sequence alignment of the sACE N-domain (SEQ ID NO: 1) with tACE

(SEQ ID NO: 2) and ACE2 (SEQ ID NO: 3). Helices are highlighted inyellow and strands in blue. The linker region is shown in pink. Helicesare numbered sequentially whether α or 3₁₀, and 3₁₀ helices are shownwith red letters. Chloride I coordinating residues are boxed in darkblue, chloride II in black, and the zinc binding motif in red.

Table 1. Crystallographic Data

Table 2. Active site residues that differ between the N (SEQ ID NO:1)and C domains (C domain numbering is as for tACE (SEQ ID NO: 2)).

Table A (SEQ ID NOs: 5 and 6). Structure co-ordinates of N-domain of ACEprotein.

Table B (SEQ ID NOs: 7 and 8). Structure co-ordinates of N-domain of ACEprotein complexed with lisinopril.

The invention will now be further described by way of Examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention.

EXAMPLES Example 1 Materials & Methods

Construction of Expression Vectors An N-domain (SEQ ID NO: 4) construct(D629) cloned into the vector pECE (a kind gift from Sergei Danilov) andencoding the first 629 residues of somatic ACE, was subcloned intopBlueScript using the restriction endonucleases XbaI and EcoRI. Thefragment was then sequenced to verify that the correct fragment wascloned. D629 was then again subcloned into pcDNA3.1(+) using the samerestriction enzymes. This N-domain (SEQ ID NO: 4) construct was thenintroduced into the CHO cell glutamine synthetase (GS) expression vectorpEE14 using the restriction endonucleases HindIII and XbaI. The identityof the construct and its correct orientation was confirmed viarestriction enzyme digests and further sequencing. The new expressionvector was termed pEE14 N-domain.Cell Culture and Transfections

CHO-K1 cells were co-transfected with pEE14-N-domain (SEQ ID NO: 4) andpSV2neo (10:1). Geneticin (G418) (Sigma) resistant clones expressingsoluble N-domain (SEQ ID NO: 4, residues 1-629) were further selectedfor resistance to methionine sulfoximine (MSX) (Yu et al., 1997). Cellsstably expressing the N-domain (SEQ ID NO: 1) were grown in GlasgowMinimum Essential Medium (GMEM) supplemented with 10% dialysed foetalcalf serum (Gibco BRL) and 20 μM MSX. When confluent growth medium waschanged to 1% dialysed FCS, 0.05% albumax I (Gibco BRL), 20 μM MSX and1.5 mM N-butyldeoxynojirimycin (NB-DNJ) (Toronto Research ChemicalsInc.B691000 Lot 14-EG-91-1 and B691000 12-Cf-146-2).

Construct Purification

Medium containing recombinantly expressed soluble N-domain (N-domain(SEQ ID NO: 4, residues 1-629) of ACE protein) was harvested andpurified in tandem over a protein-G Agarose (Sigma) column followed byan N-domain specific monoclonal antibody (5C5) protein G Agarose column.N-domain was eluted with 50 mM ethanolamine pH 11.5. The eluted solutionwas then dialysed against 5 mM HEPES pH 7.5, 0.1 mM PMSF andconcentrated in a 30 kDa Amicon concentrator at 1000-2000 g and 4° C. toa concentration of 10 mg/ml, and stored at 4° C.

Western Blot Analysis

The N-domain (SEQ ID NO: 4, residues 1-629) of ACE protein was detectedand its purity assessed by Western Blot analysis using 10% SDS-PAGE andtransferred on nitrocellulose membrane (Hybond-C, Amersham). Themembrane is probed with N-domain specific monoclonal antibody (5C5) anddeveloped with using the ECP chemiluminescent kit (Amersham) andvisualised on autoradiographic film according to the manufacturer'sinstructions.

Crystallisation and X-Ray Diffraction Data Collection

1 μl of the purified N-domain (SEQ ID NO: 4, residues 1-629) of ACEprotein at 4 mg/ml was mixed with 1 μl reservoir solution (0.2M lithiumsulphate, 0.1M sodium acetate, pH 4.9, 10 μM zinc sulphate, 15%polyethylene glycol 4000) and suspended above the reservoir as a hangingdrop at 16° C. Crystals grew within 1-2 weeks.

A single crystal was cryocooled (100 K) using reservoir solution plus25% glycerol as a cryoprotectant. Diffraction data to 3.0 Å werecollected on station PX14.1 of the synchrotron radiation source(Daresbury, U.K.) using a Quantum 4 charge-coupled-device detector (AreaDetection Systems, Poway, Calif.). The data were processed and scaled byusing the HKL2000 software package (HKL Research, Charlottesville, Va.)(Otwinowski, W. Oscillation data reduction program. in Proceedings ofthe CCP4 weekend 56-62 (Daresbury Laboratory, Warrington, U K, 1993).The symmetry and systematic absences were consistent with the C222₁space group (unit cell dimensions, a=101.12 Å; b=211.32 Å; and c=171.27Å) with two proteins per asymmetric unit. The crystals contained ˜54%solvent. Data reduction was carried out by using the CCP4 programTRUNCATE (CCP4. The CCP4 suite: Programs for protein crystallography.Acta Crystallogr. D50, 760-763 (1994)).

The structure of the N-domain (SEQ ID NO: 1) was solved with the programMOLREP (Vagin, A. & Teplyakov, A. An approach to multi-copy search inmolecular replacement. Acta Crystallogr. D56, 1622-1624 (2000)) using ahomology model of the N-domain (SEQ ID NO: 1) based on the tACE (SEQ IDNO: 2) structure (protein data bank entry 1O8A) as a search model.Initial refinement was performed using REFMAC Murshudov, G. N.Refinement of macromolecular structures by the maximum-likelihoodmethod. Acta Crystallogr. D53, 240-255 (1997) through the CCP4iinterface (CCP4. The CCP4 suite: Programs for protein crystallography.Acta Crystallogr. D50, 760-763 (1994)). 4% of reflections was kept asidefor Rfree calculation (Brünger, A. T. Free R value: a novel statisticalquantity for assessing the accuracy of crystal structures. Nature 355,472-475 (1992)). A large part of the C terminal region not present inthe tACE (SEQ ID NO: 2) structure could be built after the first roundof refinement using the program Coot (Emsley, P. & Cowtan, K. Coot:Model building tools for molecular graphics. Acta Crystallogr. 60,2126-2132 (2004)). Further rounds of refinement and model buildingallowed the building of the N terminus and the addition of water,carbohydrate and glycerol molecules plus an acetate ion. Finalrefinement was done using the CNS suite (Brünger, A. T. et al.Crystallography & NMR system: A new software suite for macromolecularstructure determination. Acta Crystallogr. D54, 905-921 (1998)).

Sequence alignment of the sACE N-domain (SEQ ID NO: 1) with tACE (SEQ IDNO: 2) and ACE2 (SEQ ID NO: 3) is shown in FIG. 4.

The N-Domain (SEQ ID NO: 4)/Lisinopril Inhibitor Complex—

4 mg/ml of purified N-domain (SEQ ID NO: 4, residues 1-629) wasincubated with 5 mM lisinopril for 5 hours at 4° C. 1 μl N-domain (SEQID NO: 4, residues 1-629) with lisinopril was mixed with 1 μl reservoirsolution (0.2M lithium sulphate, 0.1M sodium acetate, pH 4.9, 10 μM zincsulphate, 18% polyethylene glycol 4000) and suspended above thereservoir as a hanging drop at 16° C. Crystals grew within 1-2 weeks inthe same form as the minimally glycosylated N-domain of ACE protein.Single crystal consistent with the C222₁ space group (unit celldimensions, a=101.32 Å; b=211.90 Å; and c=171.03 Å). Diffraction data to2.8 Å were collected as for the minimally glycosylated N-domain dataset. The data were processed with MOSFLM (Leslie, A. G. W. Recentchanges to the MOSFLM package for processing film and image plate data.Joint CCP4+ESF-EAMCB Newsletter on Protein Crystallography 26(1992)),scaled with SCALA (CCP4. The CCP4 suite: Programs for proteincrystallography. Acta Crystallogr. D50, 760-763 (1994)) and datareduction carried out by TRUNCATE (CCP4. The CCP4 suite: Programs forprotein crystallography. Acta Crystallogr. D50, 760-763 (1994)).

Refinement (with 2.3% of the reflection kept for the Rfree calculation)of the minimally glycosylated N-domain structure with the CNS suite(Brünger, A. T. et al. Crystallography & NMR system: A new softwaresuite for macromolecular structure determination. Acta Crystallogr. D54,905-921 (1998)) showed clear difference density for the lisinopril,which was modelled with the program Coot (Emsley, P. & Cowtan, K. Coot:Model building tools for molecular graphics. Acta Crystallogr. 60,2126-2132 (2004)), along with a few water, carbohydrate, glycerolmolecules and an acetate ion.

The atomic coordinates have been deposited with the Protein Data Bank,www.rcsb.org, and the accession codes are 2C6F and 2C6N for theminimally glycosylated and lisinopril complexes for sACE N-domainrespectively.

All crystals are isomorphous.

Example 2 Overall Structure of the N-Domain

The N-domain (SEQ ID NO: 4, residues 1-629) was crystallised with twomolecules per asymmetric unit. The overall fold consists of a mainlyhelical secondary structure, with the same topology as tACE (SEQ ID NO:2). The N-domain has the ellipsoid shape with a central groove dividingit into two subdomains, one containing the N-terminal lid that coversthe central binding cavity. There are 27 helices, of which 18 of themare a helices, 5 are short 3 helices and 4 are mixed (Woodman, Z. L. etal., The N-domain of somatic angiotensin-converting enzyme negativelyregulates ectodomain shedding and catalytic activity. Biochem J. 389,739-744 (2005)). There are also 6 short β-strands. The structures ofboth molecules in the asymmetric are very similar with a root meansquare deviation for the Cα □atoms of 0.50 Å. Analysis of theRamachandran plot using the program PROCHECK shows that 94% of theresidues lie in the most favoured region, with none in the disallowedregion (Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thronton, J.M. PROCHECK—A program to check the stereochemical quality of proteinstructures. J. Appl. Crystallogr. 26, 283-291 (1993)). Both termini arewell ordered with all the residues (1-612) being modelled for molecule Aand only the N-terminal residue missing in molecule B.

The catalytic zinc ion was observed at the active site and one chloride,equivalent to chloride 2 of tACE (SEQ ID NO: 2), adjacent to Arg500.There are ten putative N-glycosylation sites on the human N-domain (SEQID NO: 1) protein and Fourier difference density was observed at five ofthese sites in our structure (FIG. 1(A)). Seven N-acetyl glucosamineresidues were modelled on molecule A at asparagines 25, 45, 117, 289 and480, and six on molecule B at residues 25, 45, 117 and 480. A mannoseresidue was also modelled on both molecules at the end of thedisaccharide at Asn25. Although the B-factors for these sugars are high,they are modelled on the basis of clear difference density, at least forthe ring portions. Twentyfive water molecules were modelled by visualinspection and a glycerol molecule was modelled on the surface of eachmolecule adjacent to Glu219. Three acetate molecules were also modelled,one on the surface next to the symmetry axis and one in each of theactive sites adjacent to Lys489, where a carboxyalanine was modelled inthe tACE (SEQ ID NO: 2) structure (Natesh, R., Schwager, S. L. U.,Sturrock, E. D. & Acharya, K. R. Crystal structure of the humanangiotensin-converting enzyme-lisinopril complex. Nature 421, 551-554(2003)). We attempted to model N-carboxyalanine at this position howeverthe density was smaller in the N-domain (SEQ ID NO: 1) structure.

The N-domain (SEQ ID NO: 4, residues 1-629) protein was alsocrystallised in the presence of 5 mM lisinopril, for which cleardifference density was observed upon refinement with the minimallyglycosylated N-domain (SEQ ID NO: 1) structure. The overall structure isas the minimally glycosylated N-domain, although the lack ofcompleteness in the data results in poorly defined density for the first30 residues of the N-terminus. The lisinopril is bound at the activesite in the same position and conformation as observed for tACE (SEQ IDNO: 2) (Natesh, R., Schwager, S. L. U., Sturrock, E. D. & Acharya, K. R.Crystal structure of the human angiotensin-converting enzyme-lisinoprilcomplex. Nature 421, 551-554 (2003)). Four N-acetyl glucosamine residueseach were modelled on both molecules at asparagines 25, 117, and 480.Nineteen water molecules, two glycerols and one acetate ion weremodelled. The acetate ion was modelled near the symmetry axis and thetwo the glycerols at equivalent surface positions on the two molecules,similar to as observed in the minimally glycosylated structure.

Comparison of the N and C Domains

The N- and C-domains of somatic ACE have ˜60% sequence identity andhence share the same overall topology as well as the highly conservedzinc binding motif at the active site (see figure x which will be asequence alignment). The most easily observable difference between theN- (based on SEQ ID NO: 1) and C-domains (based on tACE (SEQ ID NO: 2))when superimposed (FIG. 1(B)) is the extra length of the N-domain (SEQID NO: 1) at the N-terminus and the C terminus, which includes theinter-domain linker. The N terminus of the N-domain protein (SEQ ID NO:1), whilst having higher than average B factors, is well defined andpacks against helix 3. Also, the loop between helices 19 and 20(residues 409-417) that was not visible in the tACE (SEQ ID NO: 2)structure, is well defined. Three other flexible loops, between helices3 and 4, strands 1 and 2 and strand 6 and helix 23, show smalldifferences between the domains.

The N-domain has been observed to be activated at lower chlorideconcentrations and to a lesser extent than the C-domain (Wei, L.,Clauser, E., Alhenc-Gelas, F. & Corvol, P. The two homologous domains ofhuman angiotensin I-converting enzyme interact differently withcompetitive inhibitors. J. Biol. Chem. 267, 13389-13405 (1992)).Consistent with this is the observation of only one chloride bound tothe N-domain (SEQ ID NO: 1), rather than the two observed for tACE (SEQID NO: 2). The chloride ion is observed at the identical site aschloride II in tACE (SEQ ID NO: 2), bound between Tyr 202 and Arg 500.At the equivalent position to the tACE (SEQ ID NO: 2) chloride I site, acrucial arginine is substituted by a histidine in the N-domain (SEQ IDNO: 1). Interestingly, ACE2 (SEQ ID NO: 3), which was also only observedto bind one chloride ion in the crystal structure, binds chloride in theequivalent position to chloride I in tACE (SEQ ID NO: 2).

The Domain Arrangement of sACE

The complex kinetics of the somatic ACE catalysis is partly due to thepresence of an active site in both the N and C domains and also thepotential for interaction between them. The N- and C-domains of sACE arejoined by a linker that is susceptible to proteolysis and is assumed tobe partly flexible (Sturrock, E. D., Danilov, S. M. & Riordan, J. F.Limited proteolysis of human kidney angiotensin-converting enzyme andgeneration of catalytically active N- and C-terminal domains. Biochem.Biophys. Res. Com. 236, 16-19 (1997)). The inclusion of the linker inthe N-domain (SEQ ID NO: 1) construct allowed us to visualise thisregion for the first time. The majority of the linker (residues 602-612)was well defined in the electron density map after the first couple ofrounds of refinement and was built into the model without ambiguity. Thefirst part of the linker appears to be rigid in our model and is held inplace by a hydrogen bond between Tyr607 and Glu 161. The last fourresidues are more flexible as they have high B factors and their sidechains are not visible (FIG. 1(C)). They form a prominent surface patch,away from the core of the N-domain, with a very flexible loop that isanchored by the disulphide bond between Cys128 and Cys136 (FIG.1(C)-(D)). Although this flexible loop is on the symmetry axis, neitherits nor the linker's conformation appear to be involved in any crystalpacking interactions. Hence it is most likely that the N-terminus of theC-domain would interact with this patch, and that it could adapt itsconformation to one or more arrangements.

There is some evidence that only one domain of sACE is capable ofcatalysis at one time and that the N-domain may be a negative regulatorof the C domain Andújar-Sánchez, M., Cámara-Artigas, A. & Jara-Pérez, V.A calorimetric study of the binding of lisinopril, enalaprilat andcaptopril to angiotensin-converting enzyme. Biophys. Chem. 111, 183-189(2004); Binevski, P. V., Sizova, E. A., Pozdnev, V. F. & Kost, O. A.Evidence for the negative cooperativity of the two active sites withinbovine somatic angiotensin-converting enzyme. FEBS Lett. 550, 84-88(2003); Woodman, Z. L. et al., The N domain of somaticangiotensin-converting enzyme negatively regulates ectodomain sheddingand catalytic activity. Biochem J. 389, 739-744 (2005)). As the flexibleregion of the linker appears to be short, then it suggests that theN-domain would interact with the N-terminal lid region of the C-domain.The lid region comprise the three (largest) N terminal α-helices in boththe N- and C-domains that partly covers the substrate channel (Natesh,R., Schwager, S. L. U., Sturrock, E. D. & Acharya, K. R. Crystalstructure of the human angiotensin-converting enzyme-lisinopril complex.Nature 421, 551-554 (2003)). It is thought that a change in conformationof the lid may be necessary for entry of the substrate and might alsocontribute to the substrate specificity of the domains. The interactionof the N-domain with the C-domain lid might reduce the flexibility ofthis region to regulate the entry of substrate to the C-domain activesite or even to form a surface that partially blocked the cavityopening. By contrast, the position of the linker ensures that theC-domain is unlikely to interact with the lid region of the N-domain,although it could block the product channel.

The specific interactions between the domains are not obvious, however,particularly as the terminal three residues of the linker are notavailable in our structure. Furthermore, several side chains on thelinker and the flexible loop, which one might assume to play a part inthe interaction, are disordered and not visible. We know from thesequence, however, that the flexible loop contains a positively chargedlysine, and the linker, three negatively charged residues. These mayinteract with some of the charged residues protruding from the C-domainlid. The N- and C-domain lids have a different shape, size (due to theextra N-terminal residues) and charge distribution. These differenceshave been suggested to have a link to the substrate specificity of thetwo domains, but it seems likely that the shape and charge of theC-domain lid will also influence its interaction with the N-domainlinker.

Based on this analysis, and the conformation of the linker, we propose atentative domain arrangement for sACE. We suggest that the inter-domainlinker could pack against the lid of the C-domain, in a similar mannerto how the extra N terminal residues of the N-domain, pack against theN-domain lid. To visualise this, two N-domains (SEQ ID NO: 1) wereoverlapped with the N-terminus of one overlaying the linker of the other(allowing for the missing 3 residues), and then the C-domain (using tACE(SEQ ID NO: 2)) was superimposed (FIG. 2(A)). The initial arrangementcaused the C-domain to overlap the flexible loop, suggesting that thisloop might mould to the C-domain. Allowing free rotation around thelinker produced a model from which it was easier to observe the residuesthat could be involved in the interaction. FIG. 2(B) shows the chargedresidues on the N- and C-domains which may help form interactionsbetween them. To complicate matters further, though, there is anN-glycosylation sequon on the flexible loop of the N-domain and the toppart of the C-domain lid (FIG. 2(A)), suggesting that inter-domaininteractions and movements may be mediated or aided by sugars.

Binding of Lisinopril and Specificity

The lisinopril bound to the active site was clearly defined, despite thepoor electron density map in some other regions of the structure. Thebinding followed the same orientation as observed for tACE (SEQ ID NO:2) with the central carboxylate coordinating the zinc ion and the phenylgroup pointing up towards the lid. The active site residues and thelisinopril molecules for the two structures superimpose well (r.m.s.deviation for Cα atoms, 0.31 Å) with nearly all the lisinopril bindingresidues conserved in the N-domain (SEQ ID NO: 4). tACE Glu 162 (SEQ IDNO: 2), which interacts electrostatically with the amine on the lysylside chain, is replaced by Asp 140 in the N-domain (SEQ ID NO: 4) awayfrom the P1′ group (FIG. 3(A)). Asp 377 of tACE (SEQ ID NO: 2), whichforms water mediated interactions with the lysyl group, is also notconserved in the N-domain and is replaced by Gln355. Gln355 couldpotentially form a hydrogen bond with the lysyl group of lisinopril,although this does not appear to be the case in our structure as it isabout 4 Å away. However, it might form water mediated interactions, butwater molecules were not visible due to limited resolution of ourstructure.

A burgeoning area in the field of ACE inhibitor design is the generationof domain specific inhibitors to more precisely regulate the actions ofthe N- and C-domains of sACE. The structures of two new inhibitors havebeen published, RXP407 and RXPA380, which are highly specific for the N-and C-domains, respectively. The structural determinants of RXP470 areknown to be the N-terminal aspartate and N-acetyl groups in the P₂position and the C-terminal amide.

The difference in phosphinic peptides at the P₂ position utilises thedifferences in the N- and C-domain active site at the S₂ subsite (Table2). RXPA380 has a large hydrophobic phenyl group at this position,whereas the N-domain specific RXP407 has a charged aspartate residue andan N-terminus acetyl group. This corresponds to the exchange of aphenylalanine residue in the C-domain to Tyr 369 in the N-domain (SEQ IDNO: 4). It is probable that the RXPA380 phenyl residue would form astacking interaction with the Phe391 of tACE (SEQ ID NO: 2), whereas theaspartate or carbonyl (if the aspartate group forms electrostaticinteractions with Arg500) of RXP407 would interact with the hydroxylgroup of Tyr369 in the N-domain (SEQ ID NO: 4). Interestingly, in boththe minimally glycosylated and lisinopril-bound N-domain (SEQ ID NO: 4)structure, a patch of difference density was observed adjacent toTyr369. This was modelled as a water molecule in the minimallyglycosylated structure and a cluster of 2-3 water molecules in thelisinopril-bound structure as it was not possible to tell if it was alarger ligand, e.g. from the crystallisation solution, at thisresolution. It is possible that these water molecules in some way mimicthe interactions the N-domain (SEQ ID NO: 1) would have with extendedinhibitors such as RXP407. Indeed, RXP407 can be modelled to make asimilar interaction with Tyr369 (FIG. 3(B)).

In our structure of the N-domain (SEQ ID NO: 4) protein, the triad ofresidues (Gln259, Lys489, Tyr498), which interact with the carboxylgroup of lisinopril, are best placed to interact with the C terminalamide. However, this triad in conserved in the C-domain and the residuesthat differ between the N- and C-domains are all located further downthe cavity. These include Ser357 and Thr358 (both valine in theC-domain) and a patch of acidic residues (FIG. 3b ). Although theC-domain also has a patch of 2, rather than 3 acidic residues, they areeither different in length or position (Table 2). These residues adoptrandom conformations in our structure, which lack partner/s in the S₂subsite for them to interact with, but could possibly extend towards theactive site and interact in the presence of RXP407. Although theresolution of our structure hinders a more detailed analysis of theactive site, the structural details presented here will aid futurerational design of domain specific ACE inhibitors.

In a further aspect, the present invention relates to a compositioncomprising the N-domain of ACE protein in a crystalline form.

In a further aspect, the present invention relates to a scalable 3Dmodel of the N-domain of ACE protein having at least a portion of thestructural co-ordinates set forth in Table A (SEQ ID NOs: 5 and 6) orTable B (SEQ ID NOs: 7 and 8).

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

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TABLE 1 Crystallographic Data Native Lisinopril complex Resolutionrange, Å 48.5-3.0 50-2.8 No. of reflections measured 70398 160032 No. ofunique reflections 36858 40318 I/□(I) (outer shell*) 9.0 (2.2) 9.2 (1.3)Completeness (outer shell*), % 99.8 (99.7) 89.0 (57.3) Rsymm (outershell*), % 14.1 (59.5) 12.6 (52.8) Rcryst, % 22.5 29.8 Rfree, % 27.431.4 Average temperature factor Å² Protein (mol A/mol B) 35.4/38.539.3/40.2 Carbohydrate (mol A/mol B) 71.8/74.6 71.8/69.8 Ligand48.8/43.2 Solvent [no. of water molecules] 24.5 [25]   18.3 [16]   Zn²⁺ion/Cl²⁻ ion 31.6/40.1 39.3/35.3 RMSD from ideal values Bond lengths, Å0.02 0.017 Bond angles, ° 1.7 2.4 RMSD, root-mean-square deviation.Outer shell, 3.11-3.00 Å and 2.95-2.8 Å for the native data andlisinopril complex respectively. Rfree calculation used 4% and 2.3% ofthe reflections for the native data and lisinopril complex respectively.

TABLE 2 Active site residues that differ between the N- (SEQ ID NO: 1)and C domains (C-domain numbering is as for tACE). (SEQ ID NO: 2)). S₁tACE/ V518 C domain N domain T496 S₂ tACE/ N70 E143 S516 A63 Y62 F391V81 Y69 C domain N domain D43 S119 N494 V36 S35 Y369 N54 H42 S₁′ tACE/E162 N374 E376 D377 V380 N277 S284 C domain N domain D140 T352 D354 N355T358 D255 E262 S₂′ tACE/ D453 S284 V380 V379 E376 C domain N domain E431D262 T358 S357 D354

The invention claimed is:
 1. A crystal of an N-Domain of ACE proteinwherein the crystal belongs to the space group C222₁, or wherein thecrystal has the unit cell dimensions: a=101.12 Å, b=211.32 Å, c=171.27Å, where in the N-domain consists of amino acids 1-629 of SEQ ID NO: 4.2. A crystal according to claim 1 wherein the N-domain of ACE protein isglycosylated with less than ten sugar moieties at amino acids 25, 45,117, 289 and 480 of SEQ ID NO:
 4. 3. A crystal according to claim 1wherein the crystal further comprises a ligand bound to the N domain ofthe ACE protein.
 4. A crystal according to claim 3 wherein the ligand isbound to the N-domain of the ACE protein by contacting one or moreresidues of the N-domain of the ACE protein selected from: Gln259,Lys489, Tyr498 of SEQ ID NO:
 4. 5. A crystal according to claim 3wherein the ligand is an inhibitor of the N-domain of ACE protein.
 6. Acrystal according to claim 5 wherein the inhibitor of ACE is lisinopril,captopril or a derivative thereof.